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PMID:26265 | [Psychological determinants of motivation in adult education (author's transl)]. | The psychology of motivation in adult education has been mostly confined to the assessment of superficial causes for attending courses and seminars. The findings were not integrated in a ced in a conclusive theory of motivation elaborated for instance by American and German psychologists. Besides that the verbalized "motives" to attend courses were hardly correlated with other psychological and social context variables. An investigation of the relationship between verbalized "motives" to attend courses, certain personality dimensions (Freiburg Personality Inventory) and some aspects of the "life space" (occupational and non-occupatoinal) revealed that the motivation to participate in adult education is embedded in the context of psychological and socio-demographic factors and that the motive structure depends on the relevance of life-long learning, for example, to keep or improve the occupational status or to meet people involved in creative activities. | [Psychological determinants of motivation in adult education (author's transl)]. The psychology of motivation in adult education has been mostly confined to the assessment of superficial causes for attending courses and seminars. The findings were not integrated in a ced in a conclusive theory of motivation elaborated for instance by American and German psychologists. Besides that the verbalized "motives" to attend courses were hardly correlated with other psychological and social context variables. An investigation of the relationship between verbalized "motives" to attend courses, certain personality dimensions (Freiburg Personality Inventory) and some aspects of the "life space" (occupational and non-occupatoinal) revealed that the motivation to participate in adult education is embedded in the context of psychological and socio-demographic factors and that the motive structure depends on the relevance of life-long learning, for example, to keep or improve the occupational status or to meet people involved in creative activities. | [
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PMID:26266 | [Vocational training courses for personell in old age work--a problem- and participants-oriented education approach (author's transl)]. | Working definitions of problem-oriented and participants-oriented approach in adult education. Planning problems in terms of needs and expectations of participants as central foci of approach; in terms of new role requirements requirements of learners and teachers; in terms of new objectives of learning such as behavioral changes as compared to information gathering. Report on three examples of courses for social workers and nursing personnell. | [Vocational training courses for personell in old age work--a problem- and participants-oriented education approach (author's transl)]. Working definitions of problem-oriented and participants-oriented approach in adult education. Planning problems in terms of needs and expectations of participants as central foci of approach; in terms of new role requirements requirements of learners and teachers; in terms of new objectives of learning such as behavioral changes as compared to information gathering. Report on three examples of courses for social workers and nursing personnell. | [
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PMID:26267 | [Continuous education for people who work with the aged, especially for people who run homes for the aged (author's transl)]. | In accordance with Paolo Freire learning is not only an intake of knowledge, but it is reflection of one's own life situation and finding of new solutions. The consequence for continuous education of people who run homes for the aged is that the process of learning has to be connected with the practical experience of the participants. The long termed course of continuous education for directors of homes for the aged therefore always starts with a definition of their tasks. That leads to a definition of learning goals, which will be approached by working out and following a planned schedule for the whole course. | [Continuous education for people who work with the aged, especially for people who run homes for the aged (author's transl)]. In accordance with Paolo Freire learning is not only an intake of knowledge, but it is reflection of one's own life situation and finding of new solutions. The consequence for continuous education of people who run homes for the aged is that the process of learning has to be connected with the practical experience of the participants. The long termed course of continuous education for directors of homes for the aged therefore always starts with a definition of their tasks. That leads to a definition of learning goals, which will be approached by working out and following a planned schedule for the whole course. | [
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PMID:26268 | [Continuing education for social workers in the field of gerontology (author's transl)]. | This article reports on a first attempt to integrate advanced training programs for social workers in the field of psycho-social gerontology into university curricula. The report concentrates on experience and problems to transmit and translate gerontological research results for the use of practitioners. Especially effective proved the integration of social workers as "experts-in-practice" into open-curricular-learning and their participation in application oriented research. | [Continuing education for social workers in the field of gerontology (author's transl)]. This article reports on a first attempt to integrate advanced training programs for social workers in the field of psycho-social gerontology into university curricula. The report concentrates on experience and problems to transmit and translate gerontological research results for the use of practitioners. Especially effective proved the integration of social workers as "experts-in-practice" into open-curricular-learning and their participation in application oriented research. | [
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PMID:26269 | [Activating help--aims and limits (author's transl)]. | The publication attempts to analyze current work directed at aiding and encouraging aged people in the Federal Republic of Germany, seen from the viewpoint of the various welfare associations, and hense from a practical angle. The considerable contrast between aims, available knowledge, and realization, is made clear by discussing individual problem fields while presenting a catalog of relevant and necessary measures. The paper also describes the requirements training and continuing education, as well as the need for research based on practice and yielding results which are relevant in the everyday life of the aged. | [Activating help--aims and limits (author's transl)]. The publication attempts to analyze current work directed at aiding and encouraging aged people in the Federal Republic of Germany, seen from the viewpoint of the various welfare associations, and hense from a practical angle. The considerable contrast between aims, available knowledge, and realization, is made clear by discussing individual problem fields while presenting a catalog of relevant and necessary measures. The paper also describes the requirements training and continuing education, as well as the need for research based on practice and yielding results which are relevant in the everyday life of the aged. | [
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PMID:26271 | [Ageing and geriatric agents in the view of experimental gerontology (authors transl)]. | Applying the research methods used in natural sciences and medicine and the concept of bionomy, it has been attempted to make basic statements about the process of aging. 140 geriatric agents have been examined with reference to the medications for their use, their connection with the aging process and particularly their effect on vitality. Finally it has been pointed out that test procedures for age parameters in experimental and clinical gerontology, which can be used in assessing geriatric agents, are in existence. | [Ageing and geriatric agents in the view of experimental gerontology (authors transl)]. Applying the research methods used in natural sciences and medicine and the concept of bionomy, it has been attempted to make basic statements about the process of aging. 140 geriatric agents have been examined with reference to the medications for their use, their connection with the aging process and particularly their effect on vitality. Finally it has been pointed out that test procedures for age parameters in experimental and clinical gerontology, which can be used in assessing geriatric agents, are in existence. | [
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PMID:26272 | [Gerontological investigations of the nucleic acid metabolism in the rat. II. DNA repair capacity in different ages (authors transl)]. | We tried to develop a model of in-vivo investigations into the DNA repair capacity of rats and, by means of this model, to determine the effect of age. To this end the DNA repair capacity was determined after gamma-irradiation with 3 groups of male white Sprague-Dawley rats aged 6, 18-20 and 32-34 months. The organs liver, kidney, heart and spleen were used for this investigation. The relative repair (RR) showed a discontinuous course both in the 4 organs, and in the 3 different age groups. We define "relative repair" as the relationship between the specific activity of DNA decomposition products and the specific activity of DNA in an irradiated test group, as compared with an unirradiated control group. In all age groups the course was similar in the liver and the spleen, especially in the young rats, as well as in the heart and the kidney. Age differences were seen to be significant in all 4 organs at particular intervals after the irradiation, which means that the maximum relative repair occurs at different times. The integration over the whole course of the investigation shows a distinct reduction in the relative repair with increasing age in all the investigated organs except the spleen, where we could not see a difference between the 2 older groups. | [Gerontological investigations of the nucleic acid metabolism in the rat. II. DNA repair capacity in different ages (authors transl)]. We tried to develop a model of in-vivo investigations into the DNA repair capacity of rats and, by means of this model, to determine the effect of age. To this end the DNA repair capacity was determined after gamma-irradiation with 3 groups of male white Sprague-Dawley rats aged 6, 18-20 and 32-34 months. The organs liver, kidney, heart and spleen were used for this investigation. The relative repair (RR) showed a discontinuous course both in the 4 organs, and in the 3 different age groups. We define "relative repair" as the relationship between the specific activity of DNA decomposition products and the specific activity of DNA in an irradiated test group, as compared with an unirradiated control group. In all age groups the course was similar in the liver and the spleen, especially in the young rats, as well as in the heart and the kidney. Age differences were seen to be significant in all 4 organs at particular intervals after the irradiation, which means that the maximum relative repair occurs at different times. The integration over the whole course of the investigation shows a distinct reduction in the relative repair with increasing age in all the investigated organs except the spleen, where we could not see a difference between the 2 older groups. | [
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PMID:26273 | [Tensibility measurements on the rat's aorta III. Analysis of longitudinal and transversal extension as related to age (authors transl)]. | Stress-strain diagrams of standardizised longitudinal and transversal stripes of the thoracic aorta of 125 male Sprague-Dawley rats aged 9, 15, 24 and 30 months were recorded by an electromechanical instrument. The stripes were subjected to three successive extension-relaxation cycles. The relaxation curves of the third cycle were approximated to the functions y = a + bx and y = cxd (longitudinal stripes) and y = mxf and y = gxh (transversal stripes) respectively. The parameters could be interpreted as measures for structural and functional properties of elastic and collagenous fibers. The age changes of the curve parameters led to the following conclusions concerning age-dependent functional alterations of the aorta: The emphasis can be placed upon the increasing resistance counteracting the extension occuring with great stroke volumes. This may lead to the reduction of the capacity of the air chamber with great stroke volumes. These phenomena seem to be mainly caused by an increase of the pitch of the spiral of the collagenic fiber and the increase of the amount and/or the stability of the collagen. Age-related alterations of the elastin and of the net structure formed by the fibers influence also the distensibility at smaller extensions but seem to be less important. Therefore, the structural alterations of the aorta with age will affect the function in the first place at large stroke volumes and not be very obvious at a basic heart performance. | [Tensibility measurements on the rat's aorta III. Analysis of longitudinal and transversal extension as related to age (authors transl)]. Stress-strain diagrams of standardizised longitudinal and transversal stripes of the thoracic aorta of 125 male Sprague-Dawley rats aged 9, 15, 24 and 30 months were recorded by an electromechanical instrument. The stripes were subjected to three successive extension-relaxation cycles. The relaxation curves of the third cycle were approximated to the functions y = a + bx and y = cxd (longitudinal stripes) and y = mxf and y = gxh (transversal stripes) respectively. The parameters could be interpreted as measures for structural and functional properties of elastic and collagenous fibers. The age changes of the curve parameters led to the following conclusions concerning age-dependent functional alterations of the aorta: The emphasis can be placed upon the increasing resistance counteracting the extension occuring with great stroke volumes. This may lead to the reduction of the capacity of the air chamber with great stroke volumes. These phenomena seem to be mainly caused by an increase of the pitch of the spiral of the collagenic fiber and the increase of the amount and/or the stability of the collagen. Age-related alterations of the elastin and of the net structure formed by the fibers influence also the distensibility at smaller extensions but seem to be less important. Therefore, the structural alterations of the aorta with age will affect the function in the first place at large stroke volumes and not be very obvious at a basic heart performance. | [
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PMID:26274 | [The motor activity as an age parameter of the rat (authors transl)]. | The motor activity as an behavioural parameter provides information about the functional state of the organism as a whole. Therefore it is an important age parameter. The results of activity measurements, however, depend strongly on the method of registration. Using 3 groups of male Sprague-Dawley rats aged 9, 15 and 29 months two methods have been tested: 1) An electronic recording: the rats were registrated in their normal cages on the Animex-Activity-Meter during the dark-phase in complete darkness. The activity measured by this method has been regarded as spontaneous activity. 2) A kinematographic method: the rats were registrated in a changed environment at constant light during the dark-phase. The activity assessed by this method has been regarded as reactive activity. Spontaneous and reactive activity show a different age dependence. For the use of the motor activity as an age parameter, both, spontaneous and reactive activity, should be assessed to get a better information about the ageing of the different functional levels of the systems governing the animal's behaviour. | [The motor activity as an age parameter of the rat (authors transl)]. The motor activity as an behavioural parameter provides information about the functional state of the organism as a whole. Therefore it is an important age parameter. The results of activity measurements, however, depend strongly on the method of registration. Using 3 groups of male Sprague-Dawley rats aged 9, 15 and 29 months two methods have been tested: 1) An electronic recording: the rats were registrated in their normal cages on the Animex-Activity-Meter during the dark-phase in complete darkness. The activity measured by this method has been regarded as spontaneous activity. 2) A kinematographic method: the rats were registrated in a changed environment at constant light during the dark-phase. The activity assessed by this method has been regarded as reactive activity. Spontaneous and reactive activity show a different age dependence. For the use of the motor activity as an age parameter, both, spontaneous and reactive activity, should be assessed to get a better information about the ageing of the different functional levels of the systems governing the animal's behaviour. | [
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PMID:26275 | [Time-dependent functions of the age course of age parameters of the rat (authors transl)]. | Using 136 male Sprague-Dawley rats of various ages, the ageing functions of parameters originating from different organisation levels of the organism were determined. The parameters which were investigated on different levels show a non linear time dependence which can be approximated by simple or composed exponential decay-functions or by rising logarithmic functions. The results suggest a logarithmic relation between biological and chronological age. | [Time-dependent functions of the age course of age parameters of the rat (authors transl)]. Using 136 male Sprague-Dawley rats of various ages, the ageing functions of parameters originating from different organisation levels of the organism were determined. The parameters which were investigated on different levels show a non linear time dependence which can be approximated by simple or composed exponential decay-functions or by rising logarithmic functions. The results suggest a logarithmic relation between biological and chronological age. | [
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PMID:26276 | The age-dependent effect of caffeine on the plasma noradrenaline content in white mice. | The level of noradrenaline (NA) in the plasma of white mice, determined radioenzymatically as described by Henry et al. changes with age. In 45 days old animals NA in plasma is 10.3 +/- 4.4 ng/ml, whereas in 220 days old animals it was found to be 22.7 +/- 8.8 ng/ml. In 45 days old animals NA in plasma is increased 30 min after caffeine (45 microgram/g) significantly to 109% of control level, whereas in the older animals it is raised insignificantly (14%). The observed NA increase in plasma with increasing age might be a compensatory reaction due to diminished sensibility of the postsynaptic adrenergic receptors to NA. The explanation for the missing raise in NA plasma level after caffeine in adult mice might be the following: Calcium ions are indispensable for the release of NA by nerve impulses and the stimulation of presynaptic beta-receptors of noradrenergic neurons by NA contributes to facilitations of NA release. As in older animals under physiological conditions the release of NA is higher than in young animals it can be assumed that in the older ones the NA release from sympathetic neurons stimulated by caffeine cannot be further enhanced since the calcium receptors are fully occupied by released calcium ions and the presynaptic beta-receptors by released NA molecules. | The age-dependent effect of caffeine on the plasma noradrenaline content in white mice. The level of noradrenaline (NA) in the plasma of white mice, determined radioenzymatically as described by Henry et al. changes with age. In 45 days old animals NA in plasma is 10.3 +/- 4.4 ng/ml, whereas in 220 days old animals it was found to be 22.7 +/- 8.8 ng/ml. In 45 days old animals NA in plasma is increased 30 min after caffeine (45 microgram/g) significantly to 109% of control level, whereas in the older animals it is raised insignificantly (14%). The observed NA increase in plasma with increasing age might be a compensatory reaction due to diminished sensibility of the postsynaptic adrenergic receptors to NA. The explanation for the missing raise in NA plasma level after caffeine in adult mice might be the following: Calcium ions are indispensable for the release of NA by nerve impulses and the stimulation of presynaptic beta-receptors of noradrenergic neurons by NA contributes to facilitations of NA release. As in older animals under physiological conditions the release of NA is higher than in young animals it can be assumed that in the older ones the NA release from sympathetic neurons stimulated by caffeine cannot be further enhanced since the calcium receptors are fully occupied by released calcium ions and the presynaptic beta-receptors by released NA molecules. | [
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PMID:26299 | Advantages and disadvantages of beta-adrenergic blocking drugs in hypertension. | This paper reviews the role of beta-adrenergic blocking drugs in the treatment of hypertension. A significant proportion of patients presenting with mild or moderate hypertension will respond to general measures and do not require specific drug therapy. Of the remaining patients, some will respond to treatment with a thiazide diuretic alone, and those with severe grades of hypertension will require the addition of a beta-adrenergic blocking drug, occasionally together with a vasodilator. The advantages of beta-adrenergic lbocking drugs alone or in combination and the problems associated with therapy are discussed. | Advantages and disadvantages of beta-adrenergic blocking drugs in hypertension. This paper reviews the role of beta-adrenergic blocking drugs in the treatment of hypertension. A significant proportion of patients presenting with mild or moderate hypertension will respond to general measures and do not require specific drug therapy. Of the remaining patients, some will respond to treatment with a thiazide diuretic alone, and those with severe grades of hypertension will require the addition of a beta-adrenergic blocking drug, occasionally together with a vasodilator. The advantages of beta-adrenergic lbocking drugs alone or in combination and the problems associated with therapy are discussed. | [
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PMID:26300 | [Fluorometric determination of free and total tryptophan in blood plasma]. | Free and total tryptophan is estimated in the plasma by a spectrofluorimetric method using an amplification reaction and fluorescence by transformation of tryptophan into norharman using formalin and perhydrol (fluorescence is amplified about 15 times). Free tryptophan in the plasma is separated by ultrafiltration under pressure. We verified the analytical characteristics of the method in order to obtain optimal conditions. Values frequently found in 16 blood donors in good health were 10.4 +/- 3.3 micronmol/l. for free tryptophan (average +/- ts for a risk of 5%) and 54.6 +/- 18.6 micronmol/l. for total tryptophan. | [Fluorometric determination of free and total tryptophan in blood plasma]. Free and total tryptophan is estimated in the plasma by a spectrofluorimetric method using an amplification reaction and fluorescence by transformation of tryptophan into norharman using formalin and perhydrol (fluorescence is amplified about 15 times). Free tryptophan in the plasma is separated by ultrafiltration under pressure. We verified the analytical characteristics of the method in order to obtain optimal conditions. Values frequently found in 16 blood donors in good health were 10.4 +/- 3.3 micronmol/l. for free tryptophan (average +/- ts for a risk of 5%) and 54.6 +/- 18.6 micronmol/l. for total tryptophan. | [
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PMID:26302 | Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. | In a previous study, we showed that increasing minute ventilation (VE) in rabbit lung by adding a dead space augmented pulmonary surfactant in the airspaces by a cholinergically mediated mechanism. Using the same model in the present study of 148 rabbits, we found that increasing VE augmented airspace phospholipid, the main component of surfactant, from 2.50 +/- 0.61 (mean +/- SD) mg per g of lung during normal VE to 3.15 +/- 1.22 (mean +/- SD) mg per g of lung during increased VE (P = 0.02). Both blocking beta-adrenergic receptors with propranolol or sotalol and inhibiting prostaglandin synthetase with indomethacin or sodium meclofenamate prevented the expected increase in phospholipid during increased VE (P is less than 0.05). The beta-2 agonist, terbutaline, increased phospholipid by 43 per cent during normal VE (P is less than 0.01), and propranolol blocked this increase (P is less than 0.05). Isoproterenol, arachidonic acid, prostaglandins E1, E2, F2alpha, and a cyclic endoperoxide analog of prostaglandin H2 (U-46619) injected during normal VE failed to increase phospholipid. We concluded that acetylcholine (previous study), beta-adrenergic mediators, and prostaglandins are involved in controlling alveolar surfactant during increased VE. | Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. In a previous study, we showed that increasing minute ventilation (VE) in rabbit lung by adding a dead space augmented pulmonary surfactant in the airspaces by a cholinergically mediated mechanism. Using the same model in the present study of 148 rabbits, we found that increasing VE augmented airspace phospholipid, the main component of surfactant, from 2.50 +/- 0.61 (mean +/- SD) mg per g of lung during normal VE to 3.15 +/- 1.22 (mean +/- SD) mg per g of lung during increased VE (P = 0.02). Both blocking beta-adrenergic receptors with propranolol or sotalol and inhibiting prostaglandin synthetase with indomethacin or sodium meclofenamate prevented the expected increase in phospholipid during increased VE (P is less than 0.05). The beta-2 agonist, terbutaline, increased phospholipid by 43 per cent during normal VE (P is less than 0.01), and propranolol blocked this increase (P is less than 0.05). Isoproterenol, arachidonic acid, prostaglandins E1, E2, F2alpha, and a cyclic endoperoxide analog of prostaglandin H2 (U-46619) injected during normal VE failed to increase phospholipid. We concluded that acetylcholine (previous study), beta-adrenergic mediators, and prostaglandins are involved in controlling alveolar surfactant during increased VE. | [
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PMID:26303 | [A short loading acidificacion test with calcium chloride in infants (author's transl)]. | The response of blood and kidney to orally administered CaCl2 (4.1 mEq. X Kg.) in a short acid loading was compared to that attained with standard agent NH4Cl (3.9 mEq. X Kg) in two groups of normal infants through a 6 hrs. period. Plasma pH and bicarbonate decreased significantly. Minimum urine pH was 5.0 o lesser in all infants. Decrease of urine pH was greater to CaCl2 than to NH4Cl group but the net hydrogen ion excretion by the kidney was minor. Accordingly this experience CaCl2 can replace NH4Cl as acidifying substance in a short acid loading test. Indication will be diffuse hepatic lesions rendering dangerous NH4Cl administration. | [A short loading acidificacion test with calcium chloride in infants (author's transl)]. The response of blood and kidney to orally administered CaCl2 (4.1 mEq. X Kg.) in a short acid loading was compared to that attained with standard agent NH4Cl (3.9 mEq. X Kg) in two groups of normal infants through a 6 hrs. period. Plasma pH and bicarbonate decreased significantly. Minimum urine pH was 5.0 o lesser in all infants. Decrease of urine pH was greater to CaCl2 than to NH4Cl group but the net hydrogen ion excretion by the kidney was minor. Accordingly this experience CaCl2 can replace NH4Cl as acidifying substance in a short acid loading test. Indication will be diffuse hepatic lesions rendering dangerous NH4Cl administration. | [
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PMID:26307 | [Extraction and polarimetric method of determining phenacetyl-D(-)-alpha-aminophenylacetic acid]. | Equilibrium distribution of phenacetyl-D-(--)-alpha-aminophenylacetic acid obtained on fermentative hydrolysis of D,L-aminophenylacetic acid in a two-phase system (chloroform-water) was studied within a wide range of the substance concentration in the organic phase. It was shown than in the organic phase the distributing substance formed associates. The empirical equation: y = A + Bx describing the dependence of the effective distribution of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid on its concentration in the organic phase was suggested. Coefficients A and B of the equation were determined and an equation for evaluating equilibrium concentrations in the equeous phase was suggested. On the basis of the studies an extraction-polarometric method for quantitative determination of phenacetyl-D-(--)-alpha-aminophenylacetic acid concentration was developed. The method consists of extraction of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid with chloroform, determination of the equilibrium concentration of the distributing substance in the organic phase by the polarimetric method and subsequent estimation of the equilibrium and initial concentrations of the electrolyte in the aqueous phase. | [Extraction and polarimetric method of determining phenacetyl-D(-)-alpha-aminophenylacetic acid]. Equilibrium distribution of phenacetyl-D-(--)-alpha-aminophenylacetic acid obtained on fermentative hydrolysis of D,L-aminophenylacetic acid in a two-phase system (chloroform-water) was studied within a wide range of the substance concentration in the organic phase. It was shown than in the organic phase the distributing substance formed associates. The empirical equation: y = A + Bx describing the dependence of the effective distribution of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid on its concentration in the organic phase was suggested. Coefficients A and B of the equation were determined and an equation for evaluating equilibrium concentrations in the equeous phase was suggested. On the basis of the studies an extraction-polarometric method for quantitative determination of phenacetyl-D-(--)-alpha-aminophenylacetic acid concentration was developed. The method consists of extraction of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid with chloroform, determination of the equilibrium concentration of the distributing substance in the organic phase by the polarimetric method and subsequent estimation of the equilibrium and initial concentrations of the electrolyte in the aqueous phase. | [
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PMID:26308 | Reisolation and growth conditions of bacillus agar-exedens. | Several agarolytic Bacillus strains have been isolated. The properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described. | Reisolation and growth conditions of bacillus agar-exedens. Several agarolytic Bacillus strains have been isolated. The properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described. | [
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PMID:26309 | Inhibition of light-induced pH increase and O2 evolution of marine microalgae by water-soluble components of crude and refined oils. | Light-induced alkalinization of the extracellular medium was found to be a common feature of the primary photosynthetic process of several marine microalgae. The light-induce PH increase of suspensions of whole cells was immediately and severely inhibited by a single dose of water-soluble components from crude and fuel oils. Differential effects on the rates of microalgal photosynthetic O2 evolution and cell suspension pH increase suggest different toxicity mechanisms of the water-soluble components of no. 2 fuel oil as compared with Southern Louisiana and Jay Crude oils. These short-term studies on the nature of sublethal petroleum toxicity to microalgae indicate that the primary effect may be through direct action on the energy-yielding electron transport systems. | Inhibition of light-induced pH increase and O2 evolution of marine microalgae by water-soluble components of crude and refined oils. Light-induced alkalinization of the extracellular medium was found to be a common feature of the primary photosynthetic process of several marine microalgae. The light-induce PH increase of suspensions of whole cells was immediately and severely inhibited by a single dose of water-soluble components from crude and fuel oils. Differential effects on the rates of microalgal photosynthetic O2 evolution and cell suspension pH increase suggest different toxicity mechanisms of the water-soluble components of no. 2 fuel oil as compared with Southern Louisiana and Jay Crude oils. These short-term studies on the nature of sublethal petroleum toxicity to microalgae indicate that the primary effect may be through direct action on the energy-yielding electron transport systems. | [
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PMID:26310 | Anaerobic utilization of phosphite and hypophosphite by Bacillus sp. | A Bacillus sp. capable of utilizing phosphite and hypophosphite under anaerobic conditions was isolated from Cape Canerval soil samples. The organism was isolated on a glucose-mineral salts medium with phosphate deleted. Anaerobic cultivation of this isolate resulted in decreases in the hypophosphite or phosphite concentration, increases in turbidity, cell count, and dry-cell weight, and decreases in pH and glucose concentration. The optimum hypophosphite concentration for this isolate was 60 microgram/ml, whereas the optimum phosphate concentration was greater than 1,000 microgram/ml, suggesting that higher concentrations of hypophosphite may be toxic to this isolate. Hypophosphite or phosphite utilization was accompanied by little or no detectable accumulation of phosphate in the medium, and 32P-labeled hypophosphite was incorporated into the cell as organic phosphate. When phosphate was present in the medium, the isolate failed to metabolize phosphite. In the presence of phosphite and hypophosphite, the isolate first utilized phosphite and then hypophosphite. | Anaerobic utilization of phosphite and hypophosphite by Bacillus sp. A Bacillus sp. capable of utilizing phosphite and hypophosphite under anaerobic conditions was isolated from Cape Canerval soil samples. The organism was isolated on a glucose-mineral salts medium with phosphate deleted. Anaerobic cultivation of this isolate resulted in decreases in the hypophosphite or phosphite concentration, increases in turbidity, cell count, and dry-cell weight, and decreases in pH and glucose concentration. The optimum hypophosphite concentration for this isolate was 60 microgram/ml, whereas the optimum phosphate concentration was greater than 1,000 microgram/ml, suggesting that higher concentrations of hypophosphite may be toxic to this isolate. Hypophosphite or phosphite utilization was accompanied by little or no detectable accumulation of phosphate in the medium, and 32P-labeled hypophosphite was incorporated into the cell as organic phosphate. When phosphate was present in the medium, the isolate failed to metabolize phosphite. In the presence of phosphite and hypophosphite, the isolate first utilized phosphite and then hypophosphite. | [
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PMID:26318 | Factors influencing intraoperative gastric regurgitation: a prospective random study of nasogastric tube drainage. | A prospective study was conducted to determine the incidence of "silent" gastric regurgitation and aspiration during general anesthesia in 146 patients randomized with respect to presence of a nasogastric tube. A bland dye was instilled in the stomach to serve as the determinant marker. The overall incidence of regugitation was 8.9% and of aspiration, 2.1% in spite of the uniform use of an endotracheal tube. The incidence of regurgitation was twice as high when anesthesia was given by an inexperienced anesthetist (11% vs 5.6%) and in patients without nasogastric tubes (12% vs 6%), although such differences were not statistically significant. The primary agent used, difficulty of endotracheal intubation, location of surgical incision, and duration of anesthesia did not alter the incidence of regurgitation or aspiration. No correlation was found between the detection of subclinical aspiration and the development of postoperative pulmonary complications. | Factors influencing intraoperative gastric regurgitation: a prospective random study of nasogastric tube drainage. A prospective study was conducted to determine the incidence of "silent" gastric regurgitation and aspiration during general anesthesia in 146 patients randomized with respect to presence of a nasogastric tube. A bland dye was instilled in the stomach to serve as the determinant marker. The overall incidence of regugitation was 8.9% and of aspiration, 2.1% in spite of the uniform use of an endotracheal tube. The incidence of regurgitation was twice as high when anesthesia was given by an inexperienced anesthetist (11% vs 5.6%) and in patients without nasogastric tubes (12% vs 6%), although such differences were not statistically significant. The primary agent used, difficulty of endotracheal intubation, location of surgical incision, and duration of anesthesia did not alter the incidence of regurgitation or aspiration. No correlation was found between the detection of subclinical aspiration and the development of postoperative pulmonary complications. | [
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PMID:26319 | [2-Br-2-ethyl-4-hydroxybutyramide and 2-Br-2-ethyl-4-butyrolactam as carbromal metabolites (author's transl)]. | 2-Brom-2-ethyl-4-hydroxybutyramide and 2-Brom-2-ethyl-4-butyrolactam have been isolated from the urine of patients with carbromal intoxications as well as from the urine of rat, mouse and dog after the application of either carbromal or bromdiethylacetamide and identified by means of mass-, NMR- and IR spectroscopy. | [2-Br-2-ethyl-4-hydroxybutyramide and 2-Br-2-ethyl-4-butyrolactam as carbromal metabolites (author's transl)]. 2-Brom-2-ethyl-4-hydroxybutyramide and 2-Brom-2-ethyl-4-butyrolactam have been isolated from the urine of patients with carbromal intoxications as well as from the urine of rat, mouse and dog after the application of either carbromal or bromdiethylacetamide and identified by means of mass-, NMR- and IR spectroscopy. | [
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PMID:26320 | [Cardiotoxicity of bromethylbutyramide (carbromide) (author's transl)]. | Bromethylbutyramide (Carbromide), a quantitatively important metabolite of carbromal, diminished the contractility of electrically driven and the frequency of spontaneously beating guinea-pig atria. The negative inotropic action of bromethylbutyramide occurred at concentrations which are reached in human carbromal poisoning. The cardiodepressive actions of bromethylbutyramide and carbromal may be important for the course and the ending of severe carbromal intoxications. | [Cardiotoxicity of bromethylbutyramide (carbromide) (author's transl)]. Bromethylbutyramide (Carbromide), a quantitatively important metabolite of carbromal, diminished the contractility of electrically driven and the frequency of spontaneously beating guinea-pig atria. The negative inotropic action of bromethylbutyramide occurred at concentrations which are reached in human carbromal poisoning. The cardiodepressive actions of bromethylbutyramide and carbromal may be important for the course and the ending of severe carbromal intoxications. | [
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PMID:26322 | [Re-evaluation of barbexaclone in 20 epileptic patients]. | The clinically observed results in 20 patients treated with barbexaclone for a period of 2 1/2 years are reported. Therapeutic efficiency as well as side effects are discussed. The authors draw attention to the fact that there was a concomitant improvement in the behaviour of some patients and a diminution of maintainence dosage in relation to a previously published trial. | [Re-evaluation of barbexaclone in 20 epileptic patients]. The clinically observed results in 20 patients treated with barbexaclone for a period of 2 1/2 years are reported. Therapeutic efficiency as well as side effects are discussed. The authors draw attention to the fact that there was a concomitant improvement in the behaviour of some patients and a diminution of maintainence dosage in relation to a previously published trial. | [
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PMID:26326 | Effect of orthophosphate and oxalate on the cold-induced release of calcium from sarcoplasmic reticulum preparations from rabbit skeletal muscle. | The cold-induced release of calcium from sarcoplasmic reticulum preparations from both white and red muscles of the rabbit was studied. Part of the release was due to the increase in pH of the reaction mixture with cooling. Calcium release was greatly reduced or completely prevented by the inclusion of oxalate or inorganic orthophosphate in the medium. No release occurred in 5 mM oxalate. With phosphate, the proportion of the calcium previously taken up at 23 degrees C that was released at 0 degrees C became progressively smaller as the phosphate concentration was increased. When the pH was adjusted to be the same at 0 degrees C as at 23 degrees C there was little release from white muscle preparations in 10 mM phosphate and no release when the phosphate concentration was 20 mM or more. With red muscle preparations calcium was released at higher phosphate concentrations, 8% of the amount previously taken up still being released at 50 mM phosphate and a smaller amount at 100 mM phosphate. The effects of oxalate and phosphate can be explained in terms of the reduction in free calcium concentration inside the vesicles by calcium precipitants, and a difference in the temperature coefficients of calcium inflow and outflow. | Effect of orthophosphate and oxalate on the cold-induced release of calcium from sarcoplasmic reticulum preparations from rabbit skeletal muscle. The cold-induced release of calcium from sarcoplasmic reticulum preparations from both white and red muscles of the rabbit was studied. Part of the release was due to the increase in pH of the reaction mixture with cooling. Calcium release was greatly reduced or completely prevented by the inclusion of oxalate or inorganic orthophosphate in the medium. No release occurred in 5 mM oxalate. With phosphate, the proportion of the calcium previously taken up at 23 degrees C that was released at 0 degrees C became progressively smaller as the phosphate concentration was increased. When the pH was adjusted to be the same at 0 degrees C as at 23 degrees C there was little release from white muscle preparations in 10 mM phosphate and no release when the phosphate concentration was 20 mM or more. With red muscle preparations calcium was released at higher phosphate concentrations, 8% of the amount previously taken up still being released at 50 mM phosphate and a smaller amount at 100 mM phosphate. The effects of oxalate and phosphate can be explained in terms of the reduction in free calcium concentration inside the vesicles by calcium precipitants, and a difference in the temperature coefficients of calcium inflow and outflow. | [
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PMID:26327 | Purification and properties of the pyrrolidonecarboxylate peptidase of Streptococcus faecium. | Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from Streptococcus faecium was purified by fractionation with streptomycin sulphate and ammonium sulphate, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit molecular weight of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulphate to be 42,000 +/- 1,000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free sulphydryl groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45 degrees C. The values of the Michaelis constants (Km) obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogues 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed. | Purification and properties of the pyrrolidonecarboxylate peptidase of Streptococcus faecium. Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from Streptococcus faecium was purified by fractionation with streptomycin sulphate and ammonium sulphate, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit molecular weight of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulphate to be 42,000 +/- 1,000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free sulphydryl groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45 degrees C. The values of the Michaelis constants (Km) obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogues 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed. | [
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PMID:26328 | Congenital and developmental anomalies of the genitalia of slaughtered bulls. | The genital tracts of 968 slaughtered bulls (46% of which were young post-puberal animals) were examined for defects of a congenital or developmental nature. The overall occurrence of such lesions was 7%. These comprised persistent penile frenulum (0.5%), hypospadias (0.3%), detached urethral process (0.4%), testicular hypoplasia (0.2%), cryptorchidism (0.6%), mesonephric duct abnormalities (1.1%) and bulbourethral cysts, fusion and aplasia (3.6%). Segmental aplasia of the mesonephric duct, not previously recorded in the study area, was found in 4 Shorthorn bulls (0.4%); 2 affected animals were from one herd. In 3 cases of hypospadias (2 from one herd), the urethra communicated with the ventral surface of the penis at the junction of the body and glans through a slit-like orifice. The occurrence of defects observed was generally comparable to that found in bull populations elsewhere but elevated occurrence of several defects in particular herds emphasized the need for further study. | Congenital and developmental anomalies of the genitalia of slaughtered bulls. The genital tracts of 968 slaughtered bulls (46% of which were young post-puberal animals) were examined for defects of a congenital or developmental nature. The overall occurrence of such lesions was 7%. These comprised persistent penile frenulum (0.5%), hypospadias (0.3%), detached urethral process (0.4%), testicular hypoplasia (0.2%), cryptorchidism (0.6%), mesonephric duct abnormalities (1.1%) and bulbourethral cysts, fusion and aplasia (3.6%). Segmental aplasia of the mesonephric duct, not previously recorded in the study area, was found in 4 Shorthorn bulls (0.4%); 2 affected animals were from one herd. In 3 cases of hypospadias (2 from one herd), the urethra communicated with the ventral surface of the penis at the junction of the body and glans through a slit-like orifice. The occurrence of defects observed was generally comparable to that found in bull populations elsewhere but elevated occurrence of several defects in particular herds emphasized the need for further study. | [
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PMID:26332 | Biochemical aspects of cardiac muscle differentiation. | Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase alpha) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3'-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase alpha are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase alpha under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3'-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3'-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3'-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases. | Biochemical aspects of cardiac muscle differentiation. Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase alpha) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3'-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase alpha are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase alpha under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3'-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3'-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3'-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases. | [
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PMID:26329 | On the mode of action of antianginal drugs. | The antianginal action of coronary dilating drugs seems to be related at least partly to their effect of improving the blood supply of the ischemic myocardium. No correlation was found between coronary dilating action of these drugs in the normal myocardium and in the ischemic area, however. It has been shown that coronary dilator drugs are able to produce a further dilatation of the vascular bed which is already in maximum hypoxic dilatation. The antianginal effect of adrenergic beta receptor blockade cannot be explained solely by its negative chronotropic and inotropic action. It involves a favourable redistribution of the coronary flow which is increased in the ischemic area. In addition, there is evidence for a direct cardiac metabolic effect of beta blockade which reduces the myocardial oxygen requirement and moderates the ischemic diminution of the myocardial lactate uptake independently of its action on the autonomic nervous control and other extracardiac factors as well as on contractility, heart rate and myocardial blood supply. | On the mode of action of antianginal drugs. The antianginal action of coronary dilating drugs seems to be related at least partly to their effect of improving the blood supply of the ischemic myocardium. No correlation was found between coronary dilating action of these drugs in the normal myocardium and in the ischemic area, however. It has been shown that coronary dilator drugs are able to produce a further dilatation of the vascular bed which is already in maximum hypoxic dilatation. The antianginal effect of adrenergic beta receptor blockade cannot be explained solely by its negative chronotropic and inotropic action. It involves a favourable redistribution of the coronary flow which is increased in the ischemic area. In addition, there is evidence for a direct cardiac metabolic effect of beta blockade which reduces the myocardial oxygen requirement and moderates the ischemic diminution of the myocardial lactate uptake independently of its action on the autonomic nervous control and other extracardiac factors as well as on contractility, heart rate and myocardial blood supply. | [
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PMID:26333 | Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations. | The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate. | Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations. The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate. | [
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PMID:26334 | Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase. | The u.v. difference spectra generated when methotrexate, trimethoprim or folate bind to Lactobacillus casei dihydrofolate reductase were analysed. The difference spectrum producted by methotrexate binding is shown to consist of three components: (a) one closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; (b) a pH-independent contribution corresponding to a 40 nm shift to longer wavelengths of a single absorption band of methotrexate: (c) a component arising from perturbation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of component (a) shows that pK of methotrexate is increased from 5.35 to 8.55 (+/-0.10) on binding. In contrast, folate is not protonated when bound to the enzyme at neutral pH. At pH7.5, where methotrexate is bound 2000 times more tightly than folate, one-third of the difference in binding energy between the two compounds arises from the difference in chaarge stage. A similar analysis of the difference spectra generated on trimethoprim binding demonstrates that this compound, too, shows an increase in pK on binding but only from 7.22 to 7.90 (+/-0.10), suggesting that its 2,4-diaminopyrimidine ring does not bind to the enzyme in precisely the same way as the corresponding moiety of methotrexate. | Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase. The u.v. difference spectra generated when methotrexate, trimethoprim or folate bind to Lactobacillus casei dihydrofolate reductase were analysed. The difference spectrum producted by methotrexate binding is shown to consist of three components: (a) one closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; (b) a pH-independent contribution corresponding to a 40 nm shift to longer wavelengths of a single absorption band of methotrexate: (c) a component arising from perturbation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of component (a) shows that pK of methotrexate is increased from 5.35 to 8.55 (+/-0.10) on binding. In contrast, folate is not protonated when bound to the enzyme at neutral pH. At pH7.5, where methotrexate is bound 2000 times more tightly than folate, one-third of the difference in binding energy between the two compounds arises from the difference in chaarge stage. A similar analysis of the difference spectra generated on trimethoprim binding demonstrates that this compound, too, shows an increase in pK on binding but only from 7.22 to 7.90 (+/-0.10), suggesting that its 2,4-diaminopyrimidine ring does not bind to the enzyme in precisely the same way as the corresponding moiety of methotrexate. | [
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PMID:26335 | Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system. | 1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups. | Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system. 1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups. | [
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PMID:26336 | Purification of plasminogen activators from human seminal plasma. | Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators. | Purification of plasminogen activators from human seminal plasma. Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators. | [
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PMID:26337 | Studies of an acid-induced species of purple membrane from Halobacterium halobium. | A new spectral species of the purple membrane of Halobacterium halobium has been observed below pH 3.2. The formation of this new species is temperature-dependent and is favoured by increasing temperature up to the physiological range of the organism. The rate of formation at pH 3.0 and 22 degrees C is 7.9 x 10-3s-1. The spectral distribution and temperature-dependence of the new species suggest that it may be phototransiet O, stabilized by low pH. Flash-photolytic experiments in the pH range 7.2-2.7 show a pH-dependence corresponding to the static events and are consistent with a single protonation of bacteriorhodopsin below pH 3.22. These results can also be interpreted in terms of the stabilization of phototransient O at low pH. The temperature-dependence of the formation of the acid-induced species may reflect a relationship with the phase transition of the membrane. | Studies of an acid-induced species of purple membrane from Halobacterium halobium. A new spectral species of the purple membrane of Halobacterium halobium has been observed below pH 3.2. The formation of this new species is temperature-dependent and is favoured by increasing temperature up to the physiological range of the organism. The rate of formation at pH 3.0 and 22 degrees C is 7.9 x 10-3s-1. The spectral distribution and temperature-dependence of the new species suggest that it may be phototransiet O, stabilized by low pH. Flash-photolytic experiments in the pH range 7.2-2.7 show a pH-dependence corresponding to the static events and are consistent with a single protonation of bacteriorhodopsin below pH 3.22. These results can also be interpreted in terms of the stabilization of phototransient O at low pH. The temperature-dependence of the formation of the acid-induced species may reflect a relationship with the phase transition of the membrane. | [
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PMID:26338 | Evidence for a proton/sugar symport in the yeast Rhodotorula gracilis (glutinis). | 1. The uptake of monosaccharides and polyols in the obligatory aerobic yeast Rhodotorula gracilis (glutinis) was accompanied by proton uptake. 2. The half-saturation constant of transport, KT, depended on pH, changing from about 2mM at pH 4.5 to 80mM at pH8.5 for D-xylose; this change of the effective carrier affinity was reversible. 3. The apparent dissociation constant of the monosaccharide carrier was estimated at pKa 6.75. 4. At pH8.5, when the pH gradient across the cell membrane vanished, no sugar accumulation was demonstrable. 5. The half-saturation constants of sugar uptake and H+ co-transport were very similar to each other, the latter obviously being controlled by the former. 6. The H+/sugar stoicheiometry remained constant under various physiological conditions; it amounted to one H+ ion per sugar molecule taken up. 7. The data are interpreted as a strong piece of evidence in favour of the active monosaccharide transport in R. gracilis (glutinis) being an H+-symport energized by the electrochemical gradient of H+ across the plasma membrane of the yeast. | Evidence for a proton/sugar symport in the yeast Rhodotorula gracilis (glutinis). 1. The uptake of monosaccharides and polyols in the obligatory aerobic yeast Rhodotorula gracilis (glutinis) was accompanied by proton uptake. 2. The half-saturation constant of transport, KT, depended on pH, changing from about 2mM at pH 4.5 to 80mM at pH8.5 for D-xylose; this change of the effective carrier affinity was reversible. 3. The apparent dissociation constant of the monosaccharide carrier was estimated at pKa 6.75. 4. At pH8.5, when the pH gradient across the cell membrane vanished, no sugar accumulation was demonstrable. 5. The half-saturation constants of sugar uptake and H+ co-transport were very similar to each other, the latter obviously being controlled by the former. 6. The H+/sugar stoicheiometry remained constant under various physiological conditions; it amounted to one H+ ion per sugar molecule taken up. 7. The data are interpreted as a strong piece of evidence in favour of the active monosaccharide transport in R. gracilis (glutinis) being an H+-symport energized by the electrochemical gradient of H+ across the plasma membrane of the yeast. | [
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PMID:26339 | Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis. | A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000. | Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis. A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000. | [
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PMID:26340 | Collagenolytic cathepsin activity in rabbit peritoneal polymorphonuclear leucocyte granules. | Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles. | Collagenolytic cathepsin activity in rabbit peritoneal polymorphonuclear leucocyte granules. Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles. | [
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PMID:26358 | Inorganic pyrophosphatase activity of the synovial fluid. Kinetic and clinical study. | Inorganic pyrophosphatase activity has been partially characterized in joint fluid and determined in patients with sporadic and familial chondrocalcinosis and their controls. Optimal pH was established at 3.5 and Km values were estimated. Ca-2 and Mg-2 did not affect this activity whereas orthophosphate (Pi) strongly inhibited it. In the clinical study no significant differences were found among groups. This suggests that if a pyrophosphatase defect is present it might be localized in joint tissue and not be reflected in synovial fluid. | Inorganic pyrophosphatase activity of the synovial fluid. Kinetic and clinical study. Inorganic pyrophosphatase activity has been partially characterized in joint fluid and determined in patients with sporadic and familial chondrocalcinosis and their controls. Optimal pH was established at 3.5 and Km values were estimated. Ca-2 and Mg-2 did not affect this activity whereas orthophosphate (Pi) strongly inhibited it. In the clinical study no significant differences were found among groups. This suggests that if a pyrophosphatase defect is present it might be localized in joint tissue and not be reflected in synovial fluid. | [
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PMID:26359 | The psychopharmacological properties of pinazepam, a new benzodiazepine derivative. | The pharmacological differences between the behavioural effects of 7-chloro-1-propargyl-5-phenyl-3H-1,4-benzodiazepin-2-one (pinazepam) and diazepam were investigated in rats. Pinazepam was more than twice as active as diazepam at a dose range between 1.25--10 mg/kg in reducing the conditioned emotional response (CER). Only at doses of 2.5 and 5 mg/kg prevented pinazepam the disruption of the avoidance responses induced by inverting the conditioned stimulus (CS). On the other hand pinazepam was less active than diazepam in reducing the number of avoidance responses in a conditioned avoidance situation. Neither pinazepam nor diazepam disrupted the conditioned responses in a fixed-interval operant behaviour. | The psychopharmacological properties of pinazepam, a new benzodiazepine derivative. The pharmacological differences between the behavioural effects of 7-chloro-1-propargyl-5-phenyl-3H-1,4-benzodiazepin-2-one (pinazepam) and diazepam were investigated in rats. Pinazepam was more than twice as active as diazepam at a dose range between 1.25--10 mg/kg in reducing the conditioned emotional response (CER). Only at doses of 2.5 and 5 mg/kg prevented pinazepam the disruption of the avoidance responses induced by inverting the conditioned stimulus (CS). On the other hand pinazepam was less active than diazepam in reducing the number of avoidance responses in a conditioned avoidance situation. Neither pinazepam nor diazepam disrupted the conditioned responses in a fixed-interval operant behaviour. | [
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PMID:26360 | [Comparative experimental studies in animals and humans on gastrointestinal blood loss following antirheumatic pharmacotherapy (author's transl)]. | The gastrointestinal blood loss caused by the two antirheumatic drugs acetyl salicylic acid (ASA) and 4-acetamidophenyl-2-acetoxybenzoate (benorilate, Benortan) was compared in experimental animals and humans by measuring the total body iron retention. In Wistar rats and humans the results indicate that the daily iron loss under ASA is significantly higher (almost by the factor 2) than that under benorilate. | [Comparative experimental studies in animals and humans on gastrointestinal blood loss following antirheumatic pharmacotherapy (author's transl)]. The gastrointestinal blood loss caused by the two antirheumatic drugs acetyl salicylic acid (ASA) and 4-acetamidophenyl-2-acetoxybenzoate (benorilate, Benortan) was compared in experimental animals and humans by measuring the total body iron retention. In Wistar rats and humans the results indicate that the daily iron loss under ASA is significantly higher (almost by the factor 2) than that under benorilate. | [
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PMID:26361 | A repeated dose comparison of the side effects of five antihistamines on objective assessments of psychomotor performance, central nervous system arousal and subjective appraisals of sleep and early morning behaviour. | The side effects of five antihistamines (chlorpheniramine maleate, mebhydrolin, clemastine hydrogen fumarate, Tavegil; ketotifen, and promethazine hydrochloride) were measured on subjective assessments of sleep and the integrity of early morning behaviour and objective assessments of complex psychomotor behaviour and central nervous system arousal. Fifty consenting volunteers each received one of the five preparations for a period of four days with the subjective effects being reported on a set of 10 cm line visual analogue scales and the objective assessments being made via a computer assisted reaction time task and critical flicker fusion thresholds. Chlorpheniramine, 4 mg t.d.s. for four days, produces a significant impairment in critical flicker fusion thresholds with respect to pretreatment baselines but none of the preparations showed any significant impairment in complex reaction time assessments. The subjective assessments of sleep and early morning hangover showed mebhydrolin and clemastine to be free from detrimental side effects, but promethazine and chlorpheniramine produce significant impairments in the integrity of early morning behaviour. | A repeated dose comparison of the side effects of five antihistamines on objective assessments of psychomotor performance, central nervous system arousal and subjective appraisals of sleep and early morning behaviour. The side effects of five antihistamines (chlorpheniramine maleate, mebhydrolin, clemastine hydrogen fumarate, Tavegil; ketotifen, and promethazine hydrochloride) were measured on subjective assessments of sleep and the integrity of early morning behaviour and objective assessments of complex psychomotor behaviour and central nervous system arousal. Fifty consenting volunteers each received one of the five preparations for a period of four days with the subjective effects being reported on a set of 10 cm line visual analogue scales and the objective assessments being made via a computer assisted reaction time task and critical flicker fusion thresholds. Chlorpheniramine, 4 mg t.d.s. for four days, produces a significant impairment in critical flicker fusion thresholds with respect to pretreatment baselines but none of the preparations showed any significant impairment in complex reaction time assessments. The subjective assessments of sleep and early morning hangover showed mebhydrolin and clemastine to be free from detrimental side effects, but promethazine and chlorpheniramine produce significant impairments in the integrity of early morning behaviour. | [
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PMID:26362 | [Capacity of clones of vaccinia virus to multiply at different temperatures and pH values]. | The study of the biological characteristics of ten clones, isolated from plaque, of vaccinia virus has made evident that two of these characteristics--the capacity to multiply at 40 degrees C and the type of CPE in animal cell cultures--can be used as genetic markers of neuropathogenicity, valued in relationship to the property to induce demyelinating encephalitis in mice injected intracerebrally or intravenously. The most neurovirulent clones demonstrated high capacity to multiply at 40 degrees C and lytic CPE; the clones, that did not have (or had at a very low level) encephalitogenic activity in mice, presented poor capacity to multiply at 40 degrees C and CPE of syncytial type. The variation of the pH of the cell culture medium enhances the difference of CPE induced by clones that had different levels of neuropathogenicity. The results are discussed in relation to their use in improving the smallpox vaccine. | [Capacity of clones of vaccinia virus to multiply at different temperatures and pH values]. The study of the biological characteristics of ten clones, isolated from plaque, of vaccinia virus has made evident that two of these characteristics--the capacity to multiply at 40 degrees C and the type of CPE in animal cell cultures--can be used as genetic markers of neuropathogenicity, valued in relationship to the property to induce demyelinating encephalitis in mice injected intracerebrally or intravenously. The most neurovirulent clones demonstrated high capacity to multiply at 40 degrees C and lytic CPE; the clones, that did not have (or had at a very low level) encephalitogenic activity in mice, presented poor capacity to multiply at 40 degrees C and CPE of syncytial type. The variation of the pH of the cell culture medium enhances the difference of CPE induced by clones that had different levels of neuropathogenicity. The results are discussed in relation to their use in improving the smallpox vaccine. | [
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PMID:26369 | Comparison of speed of onset of fazadinium, pancuronium, tubocurarine and suxamethonium. | Under light general anaesthesia the speed of onset of action of fazadinium, pancuronium, tubocurarine and suxamethonium has been assessed by measuring the decrease in the twitch height of the adductor pollicis muscle following administration of the drugs. Suxamethonium produced 95% depression of the twitch height significantly faster than any of the other drugs, while the onset of fazadinium was significantly more rapid than that of the other non-depolarizing neuromuscular blocking drugs. | Comparison of speed of onset of fazadinium, pancuronium, tubocurarine and suxamethonium. Under light general anaesthesia the speed of onset of action of fazadinium, pancuronium, tubocurarine and suxamethonium has been assessed by measuring the decrease in the twitch height of the adductor pollicis muscle following administration of the drugs. Suxamethonium produced 95% depression of the twitch height significantly faster than any of the other drugs, while the onset of fazadinium was significantly more rapid than that of the other non-depolarizing neuromuscular blocking drugs. | [
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PMID:26371 | A comparison of four beta-adrenoceptor antagonists in patients with asthma. | 1 Cardiovascular and airways response to two non-cardioselective beta-adrenoceptor blocking drugs, propranolol and pindolol (with partial agonist activity) and two cardioselective beta-adrenoceptor blocking drugs, acebutolol (with partial agonist activity) and atenolol, were compared in twelve patients with asthma. 2 All four drugs produced a significant reduction in resting pulse rate and prevented the increase in heart rate following inhaled isoprenaline (1,500 microgram). 3 Seven patients in clinical remission showed no significant bronchoconstrictor response to any of the drugs. In the remaining five patients, bronchoconstriction was greatest following propranolol (mean reduction in FEV1 26.6%) and least following atenolol (mean reduction in FEV1 6.5%). 4 The bronchodilator response to inhaled isoprenaline was blocked by propranolol and pindolol but not by acebutolol and atenolol. 5 Partial agonist activity did not appear to be clinically useful. | A comparison of four beta-adrenoceptor antagonists in patients with asthma. 1 Cardiovascular and airways response to two non-cardioselective beta-adrenoceptor blocking drugs, propranolol and pindolol (with partial agonist activity) and two cardioselective beta-adrenoceptor blocking drugs, acebutolol (with partial agonist activity) and atenolol, were compared in twelve patients with asthma. 2 All four drugs produced a significant reduction in resting pulse rate and prevented the increase in heart rate following inhaled isoprenaline (1,500 microgram). 3 Seven patients in clinical remission showed no significant bronchoconstrictor response to any of the drugs. In the remaining five patients, bronchoconstriction was greatest following propranolol (mean reduction in FEV1 26.6%) and least following atenolol (mean reduction in FEV1 6.5%). 4 The bronchodilator response to inhaled isoprenaline was blocked by propranolol and pindolol but not by acebutolol and atenolol. 5 Partial agonist activity did not appear to be clinically useful. | [
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PMID:26372 | Circulatory and alpha-adrenoceptor blocking effects of phentolamine. | 1 Intravenously administered phentolamine provoked immediate decreases in diastolic blood pressure but increases in heart rate and cardiac output. 2 These immediate circulatory effects had largely disappeared twenty minutes after administration and at this time phentolamine did not inhibit increases in blood pressure which were provoked during hand immersion in ice-cold water. 3 Log dose-response curves of noradrenaline induced increases in systolic and diastolic pressure 20 min after intravenous phentolamine were shifted to the right in a parallel manner compared with the curves before phentolamine administration. 4 It was concluded that the immediate and short acting effects induced by phentolamine are due to a non-specific vasodilator effect but in addition phentolamine causes a longer acting alpha-adrenoceptor blockade at vascular adrenoceptor sites. However, by producing both pre- and post-synaptic alpha-adrenoceptor blockade this may explain why this drug exerts only a weak antihypertensive effect. | Circulatory and alpha-adrenoceptor blocking effects of phentolamine. 1 Intravenously administered phentolamine provoked immediate decreases in diastolic blood pressure but increases in heart rate and cardiac output. 2 These immediate circulatory effects had largely disappeared twenty minutes after administration and at this time phentolamine did not inhibit increases in blood pressure which were provoked during hand immersion in ice-cold water. 3 Log dose-response curves of noradrenaline induced increases in systolic and diastolic pressure 20 min after intravenous phentolamine were shifted to the right in a parallel manner compared with the curves before phentolamine administration. 4 It was concluded that the immediate and short acting effects induced by phentolamine are due to a non-specific vasodilator effect but in addition phentolamine causes a longer acting alpha-adrenoceptor blockade at vascular adrenoceptor sites. However, by producing both pre- and post-synaptic alpha-adrenoceptor blockade this may explain why this drug exerts only a weak antihypertensive effect. | [
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PMID:26370 | The second Lilly Prize Lecture, University of Newcastle, July 1977. beta-Adrenergic receptor blockade in hypertension, past, present and future. | All beta-adrenoceptor blocking drugs that have been described share the common property of being competitive inhibitors. They differ in their associated properties, the presence or absence of cardioselectivity, membrane stabilizing activity, and partial agonist activity. Recently some beta-adrenoceptor blocking drugs have been reported which also possess alpha-adrenoceptor blocking activity. The associated properties have been used as a basis for classifying beta-adrenoceptor blocking drugs (Fitzgerald, 1969, 1972). The presence or absence of cardioselectivity is most useful for dividing beta-adrenoceptor blocking drugs. The non-selective drugs (Division I) can be further divided according to the presence or absence of intrinsic sympathomimetic activity (ISA) and membrane stabilizing activity (Fitzgerald's groups I-IV). Group I possess both membrane activity and ISA, e.g. alprenolol, oxprenolol, group II just membrane action, e.g. propanolol, group III ISA but no membrane action, e.g. pindolol. Fitzgerald placed pindolol in group I but should be placed in group III as it possesses a high degree of beta-adrenoceptor blocking potency in relation to its membrane activity (Prichard, 1974). Finally drugs in group IV have neither ISA nor membrane action, e.g. sotalol, timolol. The cardioselective drugs (Division II) can be similarly sub-divided into groups I-IV according to the presence or absence of ISA or membrane action (Fitzgerald grouped all these together as group V). Lastly there are new beta-adrenergic receptor blocking drugs which in addition have alpha- adrenergic receptor blocking properties (Division III). | The second Lilly Prize Lecture, University of Newcastle, July 1977. beta-Adrenergic receptor blockade in hypertension, past, present and future. All beta-adrenoceptor blocking drugs that have been described share the common property of being competitive inhibitors. They differ in their associated properties, the presence or absence of cardioselectivity, membrane stabilizing activity, and partial agonist activity. Recently some beta-adrenoceptor blocking drugs have been reported which also possess alpha-adrenoceptor blocking activity. The associated properties have been used as a basis for classifying beta-adrenoceptor blocking drugs (Fitzgerald, 1969, 1972). The presence or absence of cardioselectivity is most useful for dividing beta-adrenoceptor blocking drugs. The non-selective drugs (Division I) can be further divided according to the presence or absence of intrinsic sympathomimetic activity (ISA) and membrane stabilizing activity (Fitzgerald's groups I-IV). Group I possess both membrane activity and ISA, e.g. alprenolol, oxprenolol, group II just membrane action, e.g. propanolol, group III ISA but no membrane action, e.g. pindolol. Fitzgerald placed pindolol in group I but should be placed in group III as it possesses a high degree of beta-adrenoceptor blocking potency in relation to its membrane activity (Prichard, 1974). Finally drugs in group IV have neither ISA nor membrane action, e.g. sotalol, timolol. The cardioselective drugs (Division II) can be similarly sub-divided into groups I-IV according to the presence or absence of ISA or membrane action (Fitzgerald grouped all these together as group V). Lastly there are new beta-adrenergic receptor blocking drugs which in addition have alpha- adrenergic receptor blocking properties (Division III). | [
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PMID:26374 | Prevention and treatment of premature labour by drugs: review of controlled clinical trials. | This review sought evidence from 18 controlled clinical trials about the benefits of drugs (hormones, ethanol or beta-sympathomimetic agents) used to prevent premature birth. Of the 16 trials comparing drug with placebo, 13 were judged to be methodologically adequate. In eight trials, drugs were given prophylactically and in five they were given therapeutically. Two of the therapeutic trials found the drug more effective than placebo in postponing delivery. However, only one therapeutic trial and one prophylactic trial found the drug to affect favourably the outcome for the infant. The side effects of the drugs were not systematically studied. Additional clinical trials are needed to justify the use of drugs to inhibit labour. | Prevention and treatment of premature labour by drugs: review of controlled clinical trials. This review sought evidence from 18 controlled clinical trials about the benefits of drugs (hormones, ethanol or beta-sympathomimetic agents) used to prevent premature birth. Of the 16 trials comparing drug with placebo, 13 were judged to be methodologically adequate. In eight trials, drugs were given prophylactically and in five they were given therapeutically. Two of the therapeutic trials found the drug more effective than placebo in postponing delivery. However, only one therapeutic trial and one prophylactic trial found the drug to affect favourably the outcome for the infant. The side effects of the drugs were not systematically studied. Additional clinical trials are needed to justify the use of drugs to inhibit labour. | [
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PMID:26375 | Influence of pH on the efficacy of pilocarpine. | Pilocarpine 4% solutions at pH 4.1 and 5.8 were compared in a double-blind clinical trial on 24 eyes of patients with primary open-angle glaucoma. Each drug was used over a period of 1 week. No significant difference in the lowering of intraocular pressure was found, and the near-neutral solution of pilocarpine was found to be equally stable when compared to the acid solution over a 6-month period. | Influence of pH on the efficacy of pilocarpine. Pilocarpine 4% solutions at pH 4.1 and 5.8 were compared in a double-blind clinical trial on 24 eyes of patients with primary open-angle glaucoma. Each drug was used over a period of 1 week. No significant difference in the lowering of intraocular pressure was found, and the near-neutral solution of pilocarpine was found to be equally stable when compared to the acid solution over a 6-month period. | [
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PMID:26378 | Properties of trout hemoglobins reconstituted with unnatural hemes. | Native globins isolated from trout hemoglobin compoents I and IV have been reconstituted with proto-, meso-, and deuteroheme, and the spectral and functional properties of the reconstituted hemoglobins have been investigated. Equilibrium and kinetic studies allow the following conclusions. (a) The properties of the proto-reconstituted hemoglobins are very similar, or indistinguishable, from those of the native Hb's I and IV. (B) The CO binding kinetics for both proteins were found to be consistent with the equilibrium data: the overall association rate constant increases (and the autocatalytic character of the reaction decreases) in the order proto, meso, deutero. (c) A marked pH dependence of both ligand affinity and cooperativity is maintained in the reconstituted Hb's IV: at pH 6 the fractional saturation with oxygen in air (Root effect) is lower for proto- than for meso- and deutero-Hb IV. The results obtained, including partial photodissociation experiments at different pH values, can be considered, to a first approximation, consistent with the basic features of a simple two-states model. | Properties of trout hemoglobins reconstituted with unnatural hemes. Native globins isolated from trout hemoglobin compoents I and IV have been reconstituted with proto-, meso-, and deuteroheme, and the spectral and functional properties of the reconstituted hemoglobins have been investigated. Equilibrium and kinetic studies allow the following conclusions. (a) The properties of the proto-reconstituted hemoglobins are very similar, or indistinguishable, from those of the native Hb's I and IV. (B) The CO binding kinetics for both proteins were found to be consistent with the equilibrium data: the overall association rate constant increases (and the autocatalytic character of the reaction decreases) in the order proto, meso, deutero. (c) A marked pH dependence of both ligand affinity and cooperativity is maintained in the reconstituted Hb's IV: at pH 6 the fractional saturation with oxygen in air (Root effect) is lower for proto- than for meso- and deutero-Hb IV. The results obtained, including partial photodissociation experiments at different pH values, can be considered, to a first approximation, consistent with the basic features of a simple two-states model. | [
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PMID:26379 | Effect of pH on the cross-bridge arrangement in synthetic myosin filaments. | Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH. | Effect of pH on the cross-bridge arrangement in synthetic myosin filaments. Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH. | [
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PMID:26381 | Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site. | Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase. | Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site. Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase. | [
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PMID:26382 | Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies. | We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism. | Carbon-13 magnetic resonance of carboxymethylated human carbonic anhydrase B. Chemical shift and spin--lattice relaxation studies. We have previously prepared Ntau-carbosymethylhistidine-200 human carbonic anhydrase B using 90% [1-13C]bromoacetate and have observed the 13C NMR resonance of the enriched carboxylate now covalently attached in the active site. We report here chemical shift studies of the zinc-free carboxymethylated enzyme and its Co2+-substituted form, as well as relaxation studies of the resonance in the zinc enzyme at three frequencies (15.04, 25.15, and 90.5 MHz). The chemical shift and relaxation data are both consistent with the immobilization of the carboxylate at pH 8 and its approach or coordination to the zinc. The relaxation data indicate that lowering the pH to 5.5 leads to internal motion of the carboxymethyl moiety, consistent with the chemical shift evidence for the disruption of the proposed zinc--carboxylate coordination. Inhibitor binding at either pH 5.5 or 8.0 eliminates whatever internal motion might be present. The relaxation data have been interpreted using theoretical calculations on dipolar and chemical shift anisotropy contributions. The combined results indicate that the catalytic consequences of the carboxymethylation may be due to the proposed zinc--carboxylate coordination and need not result from the disruption of any role that histidine-200 might play in the catalytic mechanism. | [
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PMID:26386 | Analogous effect of protons and inositol hexaphosphate on the alteration of structure of nitrosyl fetal human hemoglobin. | We have determined the low temperature EPR spectra and room temperature ligand dissociation rate constants of human NO-hemoglobins F and A as a function of pH and inositol hexaphosphate levels in order to assess the contribution of a quaternary structural equilibrium in the two proteins to their spectral and functional properties. Our results are consistent with an increased stability of a ligated low affinity structure in the fetal protein; the functional properties of this structure appear to be essentially the same in the two hemoglobins, even though its stability relative to a high affinity conformation is different. The pH dependence of the NO dissociation constant in both adult and fetal hemoglobin can be assigned primarily to the pH-dependent equilibria of high and low affinity forms as monitored by EPR. | Analogous effect of protons and inositol hexaphosphate on the alteration of structure of nitrosyl fetal human hemoglobin. We have determined the low temperature EPR spectra and room temperature ligand dissociation rate constants of human NO-hemoglobins F and A as a function of pH and inositol hexaphosphate levels in order to assess the contribution of a quaternary structural equilibrium in the two proteins to their spectral and functional properties. Our results are consistent with an increased stability of a ligated low affinity structure in the fetal protein; the functional properties of this structure appear to be essentially the same in the two hemoglobins, even though its stability relative to a high affinity conformation is different. The pH dependence of the NO dissociation constant in both adult and fetal hemoglobin can be assigned primarily to the pH-dependent equilibria of high and low affinity forms as monitored by EPR. | [
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PMID:26387 | Spin-label studies of the sulfhydryl environment in bovine plasma albumin. 1. The N--F transition and acid expansion. | The environment of the sulfhydryl group in plasma albumin was previously characterized by employing spin-labels of varying chain lengths (Hull, H. H., Chang, R., & Kaplan, L. J. (1975) Biochim. Biophys. Acta 400, 132). It was established that the sulfhydryl is in a crevice approximately 10 A deep but this crevice was not identified further. We now report the results of titrating albumin through the acidic conformational transitions while monitoring the electron-spin resonance of the bound nitroxide. With short spin-labels a general change is observed as the pH is lowered but the N--F transition is not discernible. However, with a spin label previously shown to project to the lip on the crevice a clear N--F transition as well as the subsequent acid expansion are observed. These results indicate that the sulfhydryl is in the crevice, formed by the domains of albumin, which opens during the N--F transition. Further results indicate that bound fatty acids do not influence the integrity of the sulfhydryl environment at neutral pH. | Spin-label studies of the sulfhydryl environment in bovine plasma albumin. 1. The N--F transition and acid expansion. The environment of the sulfhydryl group in plasma albumin was previously characterized by employing spin-labels of varying chain lengths (Hull, H. H., Chang, R., & Kaplan, L. J. (1975) Biochim. Biophys. Acta 400, 132). It was established that the sulfhydryl is in a crevice approximately 10 A deep but this crevice was not identified further. We now report the results of titrating albumin through the acidic conformational transitions while monitoring the electron-spin resonance of the bound nitroxide. With short spin-labels a general change is observed as the pH is lowered but the N--F transition is not discernible. However, with a spin label previously shown to project to the lip on the crevice a clear N--F transition as well as the subsequent acid expansion are observed. These results indicate that the sulfhydryl is in the crevice, formed by the domains of albumin, which opens during the N--F transition. Further results indicate that bound fatty acids do not influence the integrity of the sulfhydryl environment at neutral pH. | [
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PMID:26388 | Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer. | Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging. | Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer. Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging. | [
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PMID:26389 | Lipoxygenation activity of purified prostaglandin-forming cyclooxygenase. | Purified cyclooxygenase, a single enzyme which catalyzes the formation of endoperoxide from arachidonic acid (20:4) in a bis(dioxygenase) reaction, is capable of oxygenating eicosadienoic acid (20:2) at C-11 in a single dioxygenase reaction. The partial oxygenation of 20:2 resembles the formation of prostaglandin from 20:4, with both oxygenation reactions exhibiting similar pH optima, substrate Km values, and cofactor effects including a need for peroxide and an absolute requirement for heme. In addition, those processes known to destroy 20:4 oxygenase activity, such as heat inactivation, inactivation with anti-inflammatory drugs, and turnover-mediated inactivation, have equally destructive effects on 20:2 oxygenase activity. Thus, both oxygenations are catalyzed by one enzyme. All of the above similarities for 20:2 and 20:4 oxygenation demonstrate that C-11 oxygenation is an integral rate-limiting step of cyclooxygenase action rather than a separate reaction resembling that of plant lipoxygenase. | Lipoxygenation activity of purified prostaglandin-forming cyclooxygenase. Purified cyclooxygenase, a single enzyme which catalyzes the formation of endoperoxide from arachidonic acid (20:4) in a bis(dioxygenase) reaction, is capable of oxygenating eicosadienoic acid (20:2) at C-11 in a single dioxygenase reaction. The partial oxygenation of 20:2 resembles the formation of prostaglandin from 20:4, with both oxygenation reactions exhibiting similar pH optima, substrate Km values, and cofactor effects including a need for peroxide and an absolute requirement for heme. In addition, those processes known to destroy 20:4 oxygenase activity, such as heat inactivation, inactivation with anti-inflammatory drugs, and turnover-mediated inactivation, have equally destructive effects on 20:2 oxygenase activity. Thus, both oxygenations are catalyzed by one enzyme. All of the above similarities for 20:2 and 20:4 oxygenation demonstrate that C-11 oxygenation is an integral rate-limiting step of cyclooxygenase action rather than a separate reaction resembling that of plant lipoxygenase. | [
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PMID:26390 | The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition. | 1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution). | The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition. 1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution). | [
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PMID:26391 | The effect of calcium on the respiratory responses of corn mitochondria. | Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage. | The effect of calcium on the respiratory responses of corn mitochondria. Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage. | [
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PMID:26392 | Modes of reduction of nitrogen in heterocysts isolated from Anabaena species. | N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I. | Modes of reduction of nitrogen in heterocysts isolated from Anabaena species. N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I. | [
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PMID:26393 | The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces. | The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes. | The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces. The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes. | [
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PMID:26394 | Effect of nucleotides on potential and pH changes across the thylakoid membrane of spinach chloroplasts. | With appropriate preparations of spinish chloroplasts we observe three distinct effects of the nucleotides: 1. An accelaration of the dark decay of the light induced 520 nm absorbance change after ATP addition. 2. An acidification of the internal space of the thylakoids after ATP addition in darkness. 3. A dark ATPase activity which is regulated by the deltapH across the membrane. We conclude that the effect of the nucleoside triphosphates on the 520 nm signal is linked to a change of the proton conductivity of the membrane, induced by the formation of a deltapH across the membrane in consequence of the dark ATPase activity. The mode of action of the nucleoside diphosphates in the presence of inorganic phosphate on the 520 nm signal is discussed. It is proposed that the effects observed are linked to the hydrolysis of the newly formed nucleoside triphosphates. | Effect of nucleotides on potential and pH changes across the thylakoid membrane of spinach chloroplasts. With appropriate preparations of spinish chloroplasts we observe three distinct effects of the nucleotides: 1. An accelaration of the dark decay of the light induced 520 nm absorbance change after ATP addition. 2. An acidification of the internal space of the thylakoids after ATP addition in darkness. 3. A dark ATPase activity which is regulated by the deltapH across the membrane. We conclude that the effect of the nucleoside triphosphates on the 520 nm signal is linked to a change of the proton conductivity of the membrane, induced by the formation of a deltapH across the membrane in consequence of the dark ATPase activity. The mode of action of the nucleoside diphosphates in the presence of inorganic phosphate on the 520 nm signal is discussed. It is proposed that the effects observed are linked to the hydrolysis of the newly formed nucleoside triphosphates. | [
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PMID:26396 | Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans. | Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction. | Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans. Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction. | [
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PMID:26397 | Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin. | In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (lambda greater than 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 leads to P380 and P380 leads to rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 leads to P380 and P380 leads to rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 leads to P380. | Kinetic study of photoregeneration process of digitonin-solubilized squid rhodopsin. In the photoregeneration process of squid rhodopsin, an intermediate has been found at neutral pH values (phosphate buffer) with a flash light (lambda greater than 540 nm). An intermediate R430, with the 11-cis retinal as chromophore, is produced from metarhodopsin in light and is converted to rhodopsin through the processes R430 leads to P380 and P380 leads to rhodopsin. The pH dependence of the velocity of the conversions suggests that processes R430 leads to P380 and P380 leads to rhodopsin involve a protolytic reaction and that the ionized group is a histidine residue of opsin. Kinetic parameters show that the largest conformational change in opsin occurs in the conversion of R430 leads to P380. | [
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PMID:26398 | The stimulation of Na+ uptake in frog skin by uranyl ions. | 1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane. | The stimulation of Na+ uptake in frog skin by uranyl ions. 1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane. | [
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PMID:26400 | Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles. | Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established. | Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles. Pyranine is shown to be a convenient and sensitive probe for reporting pH values, pHi, at the interior of anionic and at the outer surface of cationic liposomes. It is well shielded from the phospholipid headgroups by water molecules in the interior of anionic liposomes, but it is bound to the surface of cationic liposomes. Hydrogen ion concentrations outside the liposomes, 'bulk pH values', pHo, were measured by a combination electrode. While pHi = pHo for neutral, pHi less than pHo for anionic and pHi greater than pHo for cationic liposomes prepared in 5.0 . 10(-3) M phosphate buffers. pKa values for the ionization of pyranine were 7.22 +/- 0.04 and 6.00 +/- 0.05 in water and at the external surface of cationic liposomes. The surface potential for cationic liposomes containing dipalmitoyl-DL-alpha-phosphatidylcholine, cholesterol and octadecylamine in the molar ratio of 1.00 : 0.634 : 1.01, were calcuated to be +72.2 mV. Proton permeabilities were measured for single and multicompartment anionic liposomes. Transfer of anionic liposomes prepared at a given pH to a solution of different pH resulted in a pH gradient if sodium phosphate or borate were used as buffers. In the presence of sodium acetate proton equilibration is promptly established. | [
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PMID:26401 | Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes. | Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane. | Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes. Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane. | [
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PMID:26402 | Influence of (DL)-propranolol and Ca2+ on membrane potential and amino acid transport in Ehrlich ascites tumor cells. | (1) (DL)-Propranolol and Ca2+ are shown to alter the transmembrane potential difference of Ehrlich ascites tumor cells as measured by means of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide, whose fluorescent intensity changes as a function of membrane potential. (2) The changes in membrane potential elicited by these agents are dependent of the external K+ concentration in a manner which suggest that the potential changes result from a specific increase in the permeability of the plasma membrane to K+. (3) Na+-dependent amino acid transport in the presence of propranolol can be modulated by varying the external K+ concentration (K+o). The initial rate of uptake is stimulated by propranolol at low K+o and inhibited at high K+o. The change in transport rate is nearly directly proportional to the natural logarithm of [K+]o in the presence of propranolol. (4) ATP depletion of the cells by preincubation with rotenone abolishes the changes in fluorescence and amino acid uptake seen with propranolol as a function of K+o. Restoration of cellular ATP with glucose in presence of Ca2+ restores both fluorescence and amino acid transport changes which occur in response to propranolol. (5) The fluorescence changes and amino acid transport changes in response to propranolol are pH dependent, with little effect seen at pH6. (6) It is concluded that the rate of Na+-dependent amino acid uptake is a function of membrane potential and is dependent on the electrochemical potential difference for Na+. | Influence of (DL)-propranolol and Ca2+ on membrane potential and amino acid transport in Ehrlich ascites tumor cells. (1) (DL)-Propranolol and Ca2+ are shown to alter the transmembrane potential difference of Ehrlich ascites tumor cells as measured by means of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide, whose fluorescent intensity changes as a function of membrane potential. (2) The changes in membrane potential elicited by these agents are dependent of the external K+ concentration in a manner which suggest that the potential changes result from a specific increase in the permeability of the plasma membrane to K+. (3) Na+-dependent amino acid transport in the presence of propranolol can be modulated by varying the external K+ concentration (K+o). The initial rate of uptake is stimulated by propranolol at low K+o and inhibited at high K+o. The change in transport rate is nearly directly proportional to the natural logarithm of [K+]o in the presence of propranolol. (4) ATP depletion of the cells by preincubation with rotenone abolishes the changes in fluorescence and amino acid uptake seen with propranolol as a function of K+o. Restoration of cellular ATP with glucose in presence of Ca2+ restores both fluorescence and amino acid transport changes which occur in response to propranolol. (5) The fluorescence changes and amino acid transport changes in response to propranolol are pH dependent, with little effect seen at pH6. (6) It is concluded that the rate of Na+-dependent amino acid uptake is a function of membrane potential and is dependent on the electrochemical potential difference for Na+. | [
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PMID:26404 | Transport of alpha-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens. | The transport of alpha-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent Km values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for alpha-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting alpha-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using 137Cs in the presence of valinomycin. Direct attempts to demonstrate a protonsymport mechanism for alpha-aminoisobutyrate transport were negative. | Transport of alpha-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens. The transport of alpha-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent Km values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for alpha-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting alpha-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using 137Cs in the presence of valinomycin. Direct attempts to demonstrate a protonsymport mechanism for alpha-aminoisobutyrate transport were negative. | [
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PMID:26405 | How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A? | 1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA. | How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A? 1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA. | [
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PMID:26406 | Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B. | The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation. | Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B. The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation. | [
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PMID:26407 | Hydroxymethylglutaryl-coenzyme A reductase. Purification and properties of the enzyme from Fusarium oxysporum. | The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature. | Hydroxymethylglutaryl-coenzyme A reductase. Purification and properties of the enzyme from Fusarium oxysporum. The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature. | [
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PMID:26408 | Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. | Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD. | Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD. | [
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PMID:26409 | Characterization of guanylate cyclase of rod outer segments of the bovine retina. | Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process. | Characterization of guanylate cyclase of rod outer segments of the bovine retina. Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process. | [
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PMID:26410 | Isoelectric focusing of carboxypeptidase N. | Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing. | Isoelectric focusing of carboxypeptidase N. Carboxypeptidase N was partially purified on a TEAE-cellulose column and subjected to isoelectric focusing in sucrose gradient columns containing ampholine gradients of pH range 3-10 and 4-8. Activity separated into two major peaks with pI values of pH 3.8 and 4.3. Both peaks were totally converted to an active desialated enzyme with isoelectric point of pH 5.2 to 5.4. These results indicate that carboxypeptidase N is a sialoprotein with at least two forms, differing in sialic acid content, in serum. Catalytic activity is not dependent upon sialic acid but the latter may possibly influence stability since loss of activity occurred in the desialated enzyme with repeat focusing. | [
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PMID:26411 | Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12. | Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied. | Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12. Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied. | [
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PMID:26412 | Effects of thiol inhibitors on hepatic guanylate cylase activity. | Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues. | Effects of thiol inhibitors on hepatic guanylate cylase activity. Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues. | [
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PMID:26413 | Lipoxygenase-like enzyme in rat testis microsomes. | Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers. | Lipoxygenase-like enzyme in rat testis microsomes. Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers. | [
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PMID:26414 | Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys). | Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A. | Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys). Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A. | [
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PMID:26415 | A proton NMR investigation of proline-24 cis-trans isomerism in corticotropin 1-32 and related peptides. | 250 MHz 1H-NMR studies performed on aqueous solutions of corticotropin1-32, corticotropin1-24, corticotropin15-32, corticotropin20-32 and corticotropin15-24 have allowed the location and the subsequent assignment of the signals of Tyrosine-23 aromatic protons and valine-22 methyl protons. These signals are sensitive to the geometry of proline-24, clearly transcribe its isomerism and yield the ratio of the cis-trans conformers. It is concluded that for large peptides in specific cases, the proton signals of side chains can be used to probe the backbone conformation. | A proton NMR investigation of proline-24 cis-trans isomerism in corticotropin 1-32 and related peptides. 250 MHz 1H-NMR studies performed on aqueous solutions of corticotropin1-32, corticotropin1-24, corticotropin15-32, corticotropin20-32 and corticotropin15-24 have allowed the location and the subsequent assignment of the signals of Tyrosine-23 aromatic protons and valine-22 methyl protons. These signals are sensitive to the geometry of proline-24, clearly transcribe its isomerism and yield the ratio of the cis-trans conformers. It is concluded that for large peptides in specific cases, the proton signals of side chains can be used to probe the backbone conformation. | [
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PMID:26416 | Role of Bohr group salt bridges in cooperativity in hemoglobin. | Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity. | Role of Bohr group salt bridges in cooperativity in hemoglobin. Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity. | [
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PMID:26417 | Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes. | Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate. | Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes. Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate. | [
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PMID:26418 | Proton NMR study of the thermodynamics and kinetics of the acid in equilibrium base transitions in reconstituted metmyoglobins. | Optical and proton NMR pH titrations of sperm whale metmyoglobin (metMb) in its native form and reconstituted with chemically modified hemes reveal that the pKa for the acid in equilibrium base transition decreases as the heme 2,4-substituents are made more electron withdrawing. The proton NMR spectra yields resonances which are averaged over the acidic and basic forms of the protein, but still exhibit significant exchange line broadening. Analysis of this exchange contribution to the linewidth is consistent with the simple kinetic scheme metMb+H2O + OH- k2 in equilibrium k1 metMbOH + H2O with k2 = 1.3 +/- 0.5 . 10(10) M-1 . s-1 and k1 = 1.6 +/- 0.6 . 10(5) s-1 for the native protein. The effect of electron-withdrawing substituents on the heme increase k2 and decrease k1. | Proton NMR study of the thermodynamics and kinetics of the acid in equilibrium base transitions in reconstituted metmyoglobins. Optical and proton NMR pH titrations of sperm whale metmyoglobin (metMb) in its native form and reconstituted with chemically modified hemes reveal that the pKa for the acid in equilibrium base transition decreases as the heme 2,4-substituents are made more electron withdrawing. The proton NMR spectra yields resonances which are averaged over the acidic and basic forms of the protein, but still exhibit significant exchange line broadening. Analysis of this exchange contribution to the linewidth is consistent with the simple kinetic scheme metMb+H2O + OH- k2 in equilibrium k1 metMbOH + H2O with k2 = 1.3 +/- 0.5 . 10(10) M-1 . s-1 and k1 = 1.6 +/- 0.6 . 10(5) s-1 for the native protein. The effect of electron-withdrawing substituents on the heme increase k2 and decrease k1. | [
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PMID:26419 | The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis. | The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-biphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4]. In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities. The thermostability of phosphofructokinase (at 53 degrees C) increased, while that of aldolase (at 48 degrees C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52 degrees C) was also unchanged. | The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis. The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-biphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4]. In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities. The thermostability of phosphofructokinase (at 53 degrees C) increased, while that of aldolase (at 48 degrees C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52 degrees C) was also unchanged. | [
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PMID:26420 | Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain. | The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process. | Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain. The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process. | [
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PMID:26421 | Binding of polyanions to carrier ampholytes in isoelectric focusing. | The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate. | Binding of polyanions to carrier ampholytes in isoelectric focusing. The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate. | [
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] |
PMID:26422 | [Acetate kinase chromatography on agarose derivatives]. | Acetate kinase from E. coli K-12 was studied chromatographically on omega-aminoalkyl polysacharide sorbents. The dependence of the protein sorption-desorption on ionic strength and the effect of pH on the acetate kinase sorption were studied. The increase in the ionic strength caused a decrease in the amount of protein sorbed on the hexamethylenediamine- and chlorotriasinehexamethylenediamine sepharoses. On hexamethylenediamine-, octamethylenediamine- and dimethylhexamethylenediamine agaroses acetate kinase was adsorbed within the pH range of 6.5-9.0, whereas on the chlorotriasinehexamethylenediamine sepharose--at pH 6.5-8.0. The active protein was eluted at ionic strength of 0.14-0.17 M. Acetate kinase was not adsorbed on the carboxypropyonylaminohexyl sepharose within the pH range studied, i.e. 5.0-9.0 and was not adsorbed on hexamethylenediamine agarose at pH 4.0 and on chlorotriasinehexamethylenediamine sepharose--at pH 9.0. The mechanism of the enzyme-adsorbent interaction is discussed. | [Acetate kinase chromatography on agarose derivatives]. Acetate kinase from E. coli K-12 was studied chromatographically on omega-aminoalkyl polysacharide sorbents. The dependence of the protein sorption-desorption on ionic strength and the effect of pH on the acetate kinase sorption were studied. The increase in the ionic strength caused a decrease in the amount of protein sorbed on the hexamethylenediamine- and chlorotriasinehexamethylenediamine sepharoses. On hexamethylenediamine-, octamethylenediamine- and dimethylhexamethylenediamine agaroses acetate kinase was adsorbed within the pH range of 6.5-9.0, whereas on the chlorotriasinehexamethylenediamine sepharose--at pH 6.5-8.0. The active protein was eluted at ionic strength of 0.14-0.17 M. Acetate kinase was not adsorbed on the carboxypropyonylaminohexyl sepharose within the pH range studied, i.e. 5.0-9.0 and was not adsorbed on hexamethylenediamine agarose at pH 4.0 and on chlorotriasinehexamethylenediamine sepharose--at pH 9.0. The mechanism of the enzyme-adsorbent interaction is discussed. | [
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PMID:26423 | [Molecular weight and tertiary structure of transketolase from baker's yeast]. | Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5. | [Molecular weight and tertiary structure of transketolase from baker's yeast]. Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5. | [
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PMID:26424 | [Properties of the high energy phosphate bonds of triphosphoinositide]. | High heat of enzymatic hydrolysis of triphosphoinositide phosphate bonds and of high-energy phosphates is observed using microcalorimetry. Heats of hydrolysis of triphosphoinositide, ADP and ATP sharply increase with increasing pH values from 6.6 to 7.4. Heat of hydrolysis of diphosphoinositide correlates with that of low-energy phosphates, pK4 and pK5 values for triphosphoinositide are found to be 7.4 and 9.3 respectively by means of potentiometric titration deltaGo' values for diphosphoinositide and triphosphoinositide are -3.5 and -7.1 kcal/mole respectively, taking into consideration the correction for heat neutralization-ionization during hydrolysis. Rapid triphosphoinositide hydrolysis takes place in 1% aqueous pyridine solution at 100 degrees C. In contrast to diphosphoinositide and monophosphoinositide, infrared spectra of triphosphoinositide have an additional absorption band at 930 cm(-1). 31P NMR method has revealed the presence of one diester and two monoester groups in the molecule of triphosphoinositide. The differences described between triphosphoinositide and other compounds with phosphomonoester groups are suggested to be due to electrostatic nonbounded interaction of vicinal diequatorial phosphate groups. | [Properties of the high energy phosphate bonds of triphosphoinositide]. High heat of enzymatic hydrolysis of triphosphoinositide phosphate bonds and of high-energy phosphates is observed using microcalorimetry. Heats of hydrolysis of triphosphoinositide, ADP and ATP sharply increase with increasing pH values from 6.6 to 7.4. Heat of hydrolysis of diphosphoinositide correlates with that of low-energy phosphates, pK4 and pK5 values for triphosphoinositide are found to be 7.4 and 9.3 respectively by means of potentiometric titration deltaGo' values for diphosphoinositide and triphosphoinositide are -3.5 and -7.1 kcal/mole respectively, taking into consideration the correction for heat neutralization-ionization during hydrolysis. Rapid triphosphoinositide hydrolysis takes place in 1% aqueous pyridine solution at 100 degrees C. In contrast to diphosphoinositide and monophosphoinositide, infrared spectra of triphosphoinositide have an additional absorption band at 930 cm(-1). 31P NMR method has revealed the presence of one diester and two monoester groups in the molecule of triphosphoinositide. The differences described between triphosphoinositide and other compounds with phosphomonoester groups are suggested to be due to electrostatic nonbounded interaction of vicinal diequatorial phosphate groups. | [
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PMID:26425 | [Indirect immunoprecipitation by rat liver polyribosomes using antibodies to tyrosine aminotransferase]. | A fraction of rat liver polyribosomes is isolated, which in its immunochemical characteristics considerably enriched with polyribosomes capable to synthesize hydrocortisone-induced liver tyrosine aminotransferase isoenzyme. This specific polyribosome fraction was purified by immunochemical fractionation of total liver polyribosomes using indirect precipitation. The content of polyribosomes in immunoprecipitates comprise 0.4-0.8% of its initial amount (before immunochemical fractionation). The ratio of specific polyribosomes in immunoprecipitates varies from 20 to 45%, which corresponds to 25-100-fold purification. The data obtained suggest that the method of indirect precipitation can be an efficient step in the isolation procedure of individual mRNA. | [Indirect immunoprecipitation by rat liver polyribosomes using antibodies to tyrosine aminotransferase]. A fraction of rat liver polyribosomes is isolated, which in its immunochemical characteristics considerably enriched with polyribosomes capable to synthesize hydrocortisone-induced liver tyrosine aminotransferase isoenzyme. This specific polyribosome fraction was purified by immunochemical fractionation of total liver polyribosomes using indirect precipitation. The content of polyribosomes in immunoprecipitates comprise 0.4-0.8% of its initial amount (before immunochemical fractionation). The ratio of specific polyribosomes in immunoprecipitates varies from 20 to 45%, which corresponds to 25-100-fold purification. The data obtained suggest that the method of indirect precipitation can be an efficient step in the isolation procedure of individual mRNA. | [
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PMID:26426 | [Light activation of NADH and NADPH]. | Illumination of NADH and NADPH by UV-light in the absence of oxygen resulted in the reduction of ferredoxin or methyl-viologen to cation-radical and under prolonged illumination to dihydrodipyridyl. The reaction may by accompanied by triplet and singlet exitation of NADH. It was shown that hematoporphyrin in aqueous solution photosensitized the reaction of NADH oxidation by ferredoxin and methylviologen to the visible region of the spectrum. Under light excitation the redox potentials of NADH and NADPH were increased up to the level exceeding the potential of hydrogen electrode. Illumination of NADH and NADPH by UV-light in the presence of bacterial hydrogenase resulted in hydrogen evolution. The reaction of hydrogen evolution could be sensitised towards the visible region of the spectrum by chlorophyll or chloroplasts. | [Light activation of NADH and NADPH]. Illumination of NADH and NADPH by UV-light in the absence of oxygen resulted in the reduction of ferredoxin or methyl-viologen to cation-radical and under prolonged illumination to dihydrodipyridyl. The reaction may by accompanied by triplet and singlet exitation of NADH. It was shown that hematoporphyrin in aqueous solution photosensitized the reaction of NADH oxidation by ferredoxin and methylviologen to the visible region of the spectrum. Under light excitation the redox potentials of NADH and NADPH were increased up to the level exceeding the potential of hydrogen electrode. Illumination of NADH and NADPH by UV-light in the presence of bacterial hydrogenase resulted in hydrogen evolution. The reaction of hydrogen evolution could be sensitised towards the visible region of the spectrum by chlorophyll or chloroplasts. | [
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PMID:26427 | [Ribosomal proteins of pea seeds behaving like anions at pH 8.6]. | A group of proteins migrating to the anode at pH 8.6 under polyacrylamide gel electrophoresis was revealed in the total protein of non-dissociated KCl-washed pea seed ribosomes. No proteins with an isoelectric point below pH 4.2 Were found. The presence of acidic proteins in 80 S ribosomes is due to the presence of a specific set of relatively acidic proteins in the total protein of large (5 major and 10 minor components) and small (2 major and 4 minor components) subunits. The mostly acidic proteins are located in the large subunit. The acidic proteins of 60S and 40S subunits are represented by the polypeptide chains with molecular weights from 48 000 to 13 000. The acidic proteins are present in the ribosomes studied in considerably less number than the basic proteins, and the former produce a very weak staining under electrophoretic analysis as compared with the latter. The data obtained suggest that 80S ribosomes of higher plants differ from animal ribosomes by a higher content of relatively acidic proteins. | [Ribosomal proteins of pea seeds behaving like anions at pH 8.6]. A group of proteins migrating to the anode at pH 8.6 under polyacrylamide gel electrophoresis was revealed in the total protein of non-dissociated KCl-washed pea seed ribosomes. No proteins with an isoelectric point below pH 4.2 Were found. The presence of acidic proteins in 80 S ribosomes is due to the presence of a specific set of relatively acidic proteins in the total protein of large (5 major and 10 minor components) and small (2 major and 4 minor components) subunits. The mostly acidic proteins are located in the large subunit. The acidic proteins of 60S and 40S subunits are represented by the polypeptide chains with molecular weights from 48 000 to 13 000. The acidic proteins are present in the ribosomes studied in considerably less number than the basic proteins, and the former produce a very weak staining under electrophoretic analysis as compared with the latter. The data obtained suggest that 80S ribosomes of higher plants differ from animal ribosomes by a higher content of relatively acidic proteins. | [
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] |
PMID:26428 | [Effect of N-alkylimidazoles on oxidation of o-dianizidine catalyzed by horseradish peroxidase]. | Effect of a number of N-alkylimidazoles (from N-methyl to N-octylimidazole) on peroxidase oxidation of o-dianizidine at pH 8.0 is studied. Alkylimidazoles studied are found to activate the reaction under given conditions, the activation type being non-competitive KA and (alpha) values are similar for all the activators, which suggests a similar mechanism of their action. Similar KA values suppose an insignificant role of hydrophobic interactions in the binding of N-alkylimidazoles with the enzyme. | [Effect of N-alkylimidazoles on oxidation of o-dianizidine catalyzed by horseradish peroxidase]. Effect of a number of N-alkylimidazoles (from N-methyl to N-octylimidazole) on peroxidase oxidation of o-dianizidine at pH 8.0 is studied. Alkylimidazoles studied are found to activate the reaction under given conditions, the activation type being non-competitive KA and (alpha) values are similar for all the activators, which suggests a similar mechanism of their action. Similar KA values suppose an insignificant role of hydrophobic interactions in the binding of N-alkylimidazoles with the enzyme. | [
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] |
PMID:26429 | [Interaction of intracellular RNAase from Aspergillus clavatus with derivatives of its substrates]. | Using spectrophotometric and kinetic methods and also the methods of protection of Aspergillus clavatus RNAse (EC 3.1.4.23) by adenine nucleotides and their components against inactivation by means of acylation or heating, it was found that RNAse-nucleotide complex was formed by association of one enzyme molecule with one nucleotide molecule. It was also shown that all components of nucleotides (base, ribose and phosphate) take part in the formation of such complex and the removal of one of them (base or phosphate) lead to loosening of bindings of remaining fragments (ribose-5'-monophosphate, adenine) with the active site of RNAse, and to disappearance of bends within the pH range of 3.0-4.0 on the plot of pKi (5'-MP) versus pH, within the pH range of 5.5-7.0 on the plot of oKi (Ado) versus pH. The possibility of participation of associative pair RNAse imidasole groups - nucleotide phosphate groups and RNAse carboxylic group - nucleotide base in the mechanism of formation of enzyme-nucleotide (enzyme-substrate) complexes is postulated. | [Interaction of intracellular RNAase from Aspergillus clavatus with derivatives of its substrates]. Using spectrophotometric and kinetic methods and also the methods of protection of Aspergillus clavatus RNAse (EC 3.1.4.23) by adenine nucleotides and their components against inactivation by means of acylation or heating, it was found that RNAse-nucleotide complex was formed by association of one enzyme molecule with one nucleotide molecule. It was also shown that all components of nucleotides (base, ribose and phosphate) take part in the formation of such complex and the removal of one of them (base or phosphate) lead to loosening of bindings of remaining fragments (ribose-5'-monophosphate, adenine) with the active site of RNAse, and to disappearance of bends within the pH range of 3.0-4.0 on the plot of pKi (5'-MP) versus pH, within the pH range of 5.5-7.0 on the plot of oKi (Ado) versus pH. The possibility of participation of associative pair RNAse imidasole groups - nucleotide phosphate groups and RNAse carboxylic group - nucleotide base in the mechanism of formation of enzyme-nucleotide (enzyme-substrate) complexes is postulated. | [
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PMID:26430 | [Subcellular distribution and several properties of the cAMP enzyme system of phototrophic bacteria]. | In the cells of the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris the two enzymes of the cAMP system enzymes - adenylate cyclase and cAMP phosphodiesterase (PDE) exist in a soluble and membrane-bound forms. After mild disruption of the cells (sonication up to 3 min) the activity of both enzymes is found in the chromatophores. In the cells of the two types of bacteria grown under anaerobic conditions soluble adenylate cyclase is predominant. In the cells of R. rubrum the soluble form of PDE posesses higher activity, whereas in the cells of Rh. palustris a higher activity is observed in the membrane-bound form. In addition to their different localization in the cells, the PDE forms of Rh. rubrum differ in their ratios to the concentrations of hydrogen ions and bivalent metals; the latter difference, however, may be accounted for by the effect of a protein modulator of PDE. The pH optimum of membrane-bound PDE is 9.15. Soluble PDE has two activity maxima at pH 7.5 and 8.7. It is probable that similar to the animal tissue enzyme, PDE from Rh. rubrum exists in the soluble phase in at least tw forms. Close pH optima for soluble adenylate cyclase and for one of the soluble PDE forms (about 8.5) may indicate the unidirectional control of these enzymes by hydrogen ion concentration. | [Subcellular distribution and several properties of the cAMP enzyme system of phototrophic bacteria]. In the cells of the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris the two enzymes of the cAMP system enzymes - adenylate cyclase and cAMP phosphodiesterase (PDE) exist in a soluble and membrane-bound forms. After mild disruption of the cells (sonication up to 3 min) the activity of both enzymes is found in the chromatophores. In the cells of the two types of bacteria grown under anaerobic conditions soluble adenylate cyclase is predominant. In the cells of R. rubrum the soluble form of PDE posesses higher activity, whereas in the cells of Rh. palustris a higher activity is observed in the membrane-bound form. In addition to their different localization in the cells, the PDE forms of Rh. rubrum differ in their ratios to the concentrations of hydrogen ions and bivalent metals; the latter difference, however, may be accounted for by the effect of a protein modulator of PDE. The pH optimum of membrane-bound PDE is 9.15. Soluble PDE has two activity maxima at pH 7.5 and 8.7. It is probable that similar to the animal tissue enzyme, PDE from Rh. rubrum exists in the soluble phase in at least tw forms. Close pH optima for soluble adenylate cyclase and for one of the soluble PDE forms (about 8.5) may indicate the unidirectional control of these enzymes by hydrogen ion concentration. | [
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PMID:26431 | [Irreversible inactivation of Chlorella glutamine synthetase by urea]. | The effect of urea on Chlorella glutamine synthetase (E. C. 6.3.1.2) activity and tertiary structure is investigated. Urea is found to inhibit the activity of glutamine synthetase, the inhibitory effect being independent on the time. The enzyme molecule relax and changes its affinity to ammonium under the effect of urea at concentrations of 1.0-4.0 M. Higher concentrations of urea (5,0 M and more) produce a dissociation of the enzyme molecule into monomers without any intermediate forms. Monomers do not possess any synthetase and transferase activities. Substrates and cofactors do not protect the enzyme from the effect of urea and do not stimulate the emzyme reactivation and reaggregation after its dissotiation. The data obtained are discussed from the viewpoint of the regulation of Chlorella glutamine synthetase activity in vivo. | [Irreversible inactivation of Chlorella glutamine synthetase by urea]. The effect of urea on Chlorella glutamine synthetase (E. C. 6.3.1.2) activity and tertiary structure is investigated. Urea is found to inhibit the activity of glutamine synthetase, the inhibitory effect being independent on the time. The enzyme molecule relax and changes its affinity to ammonium under the effect of urea at concentrations of 1.0-4.0 M. Higher concentrations of urea (5,0 M and more) produce a dissociation of the enzyme molecule into monomers without any intermediate forms. Monomers do not possess any synthetase and transferase activities. Substrates and cofactors do not protect the enzyme from the effect of urea and do not stimulate the emzyme reactivation and reaggregation after its dissotiation. The data obtained are discussed from the viewpoint of the regulation of Chlorella glutamine synthetase activity in vivo. | [
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PMID:26432 | Influence of pH and temperature on behaviour of surfactant from human neonatal lungs. | The surface pressures of human surfactant films were measured under static conditions and under dynamic (expansion-compression) conditions. The results from both methods showed that the maximum surface pressures generated by surfactant films in vitro were strongly dependent on temperature throughout the range 29-41 degrees C, higher pressures being achieved at the lower temperatures. In contrast, increasing the pH of the subphase stepwise over the range 7.1-7.7 produced a modest fall in the maximum pressures exerted by the films. The possibility that surfactant films may behave in a similar manner in vivo is discussed. | Influence of pH and temperature on behaviour of surfactant from human neonatal lungs. The surface pressures of human surfactant films were measured under static conditions and under dynamic (expansion-compression) conditions. The results from both methods showed that the maximum surface pressures generated by surfactant films in vitro were strongly dependent on temperature throughout the range 29-41 degrees C, higher pressures being achieved at the lower temperatures. In contrast, increasing the pH of the subphase stepwise over the range 7.1-7.7 produced a modest fall in the maximum pressures exerted by the films. The possibility that surfactant films may behave in a similar manner in vivo is discussed. | [
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PMID:26433 | In vitro effects of inosine-pyruvate-phosphate on P50 values and DPG contents of fresh and stored blood from healthy neonates, symptom-free premature infants and premature infants with respiratory distress syndrome. | A study was made of the effect of a 3-hour incubation with inosine-pyruvate-phosphate (IPP) on the P50 values and 2,3-DPG contents of fresh blood and blood which had stood for 96 h, from healthy adults, term neonates, 1-2-week-old symptom-free premature infants and RDS premature infants. It was found that the P50 value and the 2,3-DPG content could be increased considerably by IPP treatment in both the fresh and the stood blood in the various groups of neonates. The question remains open as to whether the effect of IPP, in significantly improving the O2 transport in neonates, is a consequence of the 2,3-DPG alone or of some other metabolite. In RDS the stimulating effect of the IPP mixture is appreciably lower. | In vitro effects of inosine-pyruvate-phosphate on P50 values and DPG contents of fresh and stored blood from healthy neonates, symptom-free premature infants and premature infants with respiratory distress syndrome. A study was made of the effect of a 3-hour incubation with inosine-pyruvate-phosphate (IPP) on the P50 values and 2,3-DPG contents of fresh blood and blood which had stood for 96 h, from healthy adults, term neonates, 1-2-week-old symptom-free premature infants and RDS premature infants. It was found that the P50 value and the 2,3-DPG content could be increased considerably by IPP treatment in both the fresh and the stood blood in the various groups of neonates. The question remains open as to whether the effect of IPP, in significantly improving the O2 transport in neonates, is a consequence of the 2,3-DPG alone or of some other metabolite. In RDS the stimulating effect of the IPP mixture is appreciably lower. | [
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PMID:26436 | [Effect of heparin polyanion on chromatin preparations obtained in solutions of low ionic strength]. | DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b. | [Effect of heparin polyanion on chromatin preparations obtained in solutions of low ionic strength]. DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b. | [
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PMID:26437 | [Property of a heat- and acid-stable inhibitor of serine proteinases from the blood serum to inhibit lymphcyte transformation stimulated by mitogens]. | Thermo- and acid-stable serine proteases inhibitor from the rabbit blood serum (TASPI) was shown to inhibit the human peripheral blood lymphocytes transformation stimulated by phytohemagglutinin (PHA) or concanavalin A. The extent of inhibition depended on the concentration of the preparation and its specific activity. The maximal inhibition of lymphocytes proliferation constituted 50 to 70%. TASPI displayed no cytotoxic activity. Considerably more effective inhibition was demonstrated by TASPI addition to the culture medium 24 hours after the addition of PHA. The antiprotease activity of crude human serum and that inactivated under different conditions is described. The results obtained suggest the participation of TASPI in the control of biological activity of the lymphoid tissue cells. | [Property of a heat- and acid-stable inhibitor of serine proteinases from the blood serum to inhibit lymphcyte transformation stimulated by mitogens]. Thermo- and acid-stable serine proteases inhibitor from the rabbit blood serum (TASPI) was shown to inhibit the human peripheral blood lymphocytes transformation stimulated by phytohemagglutinin (PHA) or concanavalin A. The extent of inhibition depended on the concentration of the preparation and its specific activity. The maximal inhibition of lymphocytes proliferation constituted 50 to 70%. TASPI displayed no cytotoxic activity. Considerably more effective inhibition was demonstrated by TASPI addition to the culture medium 24 hours after the addition of PHA. The antiprotease activity of crude human serum and that inactivated under different conditions is described. The results obtained suggest the participation of TASPI in the control of biological activity of the lymphoid tissue cells. | [
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PMID:26443 | Effects of oxypertine on the isolated vas deferens of the rat. | 1 The isolated vas deferens of the rat was used to examine the peripheral action of oxypertine, a psychotropic-anxiolytic drug. 2 Oxypertine (4.4 X 10(-10) M to 2.6 X 10(-5) M) antagonized competitively the effects of noradrenaline (pA2 = 7.2), 5-hydroxytryptamine (pA2 = 8.6) and dopamine (pA2 = 9.8). 3 Oxypertine (8.8 X 10(-9) M to 2.6 X 10(-5) M) antagonized the effects of low concentrations of acetylcholine and enhanced the contractions elicited by high concentrations of acetylcholine. 4 The contractions evoked by transmural stimulation of the vas deferens were reduced by oxypertine. 5 Oxypertine failed to antagonize the responses to potassium chloride. 6 These findings are compared with the effects of other antidepressant drugs. | Effects of oxypertine on the isolated vas deferens of the rat. 1 The isolated vas deferens of the rat was used to examine the peripheral action of oxypertine, a psychotropic-anxiolytic drug. 2 Oxypertine (4.4 X 10(-10) M to 2.6 X 10(-5) M) antagonized competitively the effects of noradrenaline (pA2 = 7.2), 5-hydroxytryptamine (pA2 = 8.6) and dopamine (pA2 = 9.8). 3 Oxypertine (8.8 X 10(-9) M to 2.6 X 10(-5) M) antagonized the effects of low concentrations of acetylcholine and enhanced the contractions elicited by high concentrations of acetylcholine. 4 The contractions evoked by transmural stimulation of the vas deferens were reduced by oxypertine. 5 Oxypertine failed to antagonize the responses to potassium chloride. 6 These findings are compared with the effects of other antidepressant drugs. | [
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PMID:26444 | Investigation of the role of histamine in antigen-induced bronchoconstriction in the ascaris-hypersensitive dog. | 1 Aerosol administration of ascaris antigen to the airways of ascaris-hypersensitive dogs provoked increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). These changes in pulmonary mechanics were not inhibited by the histamine H1-receptor antagonists, diphenhydramine or mepyramine. 2 Increases in Rp and decreases in Cdyn induced by a histamine aerosol were markedly or totally inhibited by comparable doses of these H1-antihistamines. 3 Doses of antigen which produced pathophysiological pulmonary responses failed to produce a detectable histamine release from the cardiopulmonary system in vivo. Aerosol antigen provocation, equivalent to 5 to 9 times greater than that which produced substantial pathophysiological pulmonary responses, did cause histamine release in vivo. 4 The canine cardiopulmonary system showed only a modest ability to remove and/or degrade circulating histamine. 5 It is concluded that histamine may not play a major role in mediating the acute antigen-induced bronchoconstriction in the ascaris-hypersensitive dog. | Investigation of the role of histamine in antigen-induced bronchoconstriction in the ascaris-hypersensitive dog. 1 Aerosol administration of ascaris antigen to the airways of ascaris-hypersensitive dogs provoked increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn). These changes in pulmonary mechanics were not inhibited by the histamine H1-receptor antagonists, diphenhydramine or mepyramine. 2 Increases in Rp and decreases in Cdyn induced by a histamine aerosol were markedly or totally inhibited by comparable doses of these H1-antihistamines. 3 Doses of antigen which produced pathophysiological pulmonary responses failed to produce a detectable histamine release from the cardiopulmonary system in vivo. Aerosol antigen provocation, equivalent to 5 to 9 times greater than that which produced substantial pathophysiological pulmonary responses, did cause histamine release in vivo. 4 The canine cardiopulmonary system showed only a modest ability to remove and/or degrade circulating histamine. 5 It is concluded that histamine may not play a major role in mediating the acute antigen-induced bronchoconstriction in the ascaris-hypersensitive dog. | [
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