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PMID:26637 | Interaction of cholera toxin and toxin derivatives with lymphocytes. III. Modulating effects in vivo by cholera toxin on the graft-versus-host reactivity of lymphoid cells: suggested inhibition of suppressor cells. | The influence of cholera toxin (CT), and thus probably of cyclic AMP, on the capacity of parental lymphoid cells to elicit a graft-versus-host reaction (GVHR) was studied. Toxin-treated DBA/1 mice were used as cell donors and untreated DBA/1xC57B1/6 F1 hybrid mice as recipients, and the GVHR reactivity of the transferred cells was estimated by their ability to induce spleen enlargement or stimulation of antibody formation ('allogenic effect') in the recipients. Spleen cells from donors intravenously injected with 1 microgram CT 1-3 days earlier, gave a significantly stronger GVHR than did spleen cells of untreated mice. Choleragenoid, a toxin analog devoid of the toxin's ability to activate plasma membrane adenylate cyclase even though it binds efficiently to cells, had no effect on the GVHR-inducing capacity of the spleen cells. The enhanced GVHR by spleen cells from toxin-treated DBA/1 animals was reduced to the normal level when the donor cells were transferred along with lymphoid cells from untreated animals of the same strain. Spleen was the most powerful source of the suppressive influence. No evidence for a redistribution of suppressor cells following administration of CT was found. Spleen cells from mice syngeneic with the recipients had no suppressive effect. The results suggest that parenterally administered CT, directly or indirectly, can inhibit a cell population in spleen which normally exerts an antigen-specific suppressive regulatory influence on the development of GVHR. | Interaction of cholera toxin and toxin derivatives with lymphocytes. III. Modulating effects in vivo by cholera toxin on the graft-versus-host reactivity of lymphoid cells: suggested inhibition of suppressor cells. The influence of cholera toxin (CT), and thus probably of cyclic AMP, on the capacity of parental lymphoid cells to elicit a graft-versus-host reaction (GVHR) was studied. Toxin-treated DBA/1 mice were used as cell donors and untreated DBA/1xC57B1/6 F1 hybrid mice as recipients, and the GVHR reactivity of the transferred cells was estimated by their ability to induce spleen enlargement or stimulation of antibody formation ('allogenic effect') in the recipients. Spleen cells from donors intravenously injected with 1 microgram CT 1-3 days earlier, gave a significantly stronger GVHR than did spleen cells of untreated mice. Choleragenoid, a toxin analog devoid of the toxin's ability to activate plasma membrane adenylate cyclase even though it binds efficiently to cells, had no effect on the GVHR-inducing capacity of the spleen cells. The enhanced GVHR by spleen cells from toxin-treated DBA/1 animals was reduced to the normal level when the donor cells were transferred along with lymphoid cells from untreated animals of the same strain. Spleen was the most powerful source of the suppressive influence. No evidence for a redistribution of suppressor cells following administration of CT was found. Spleen cells from mice syngeneic with the recipients had no suppressive effect. The results suggest that parenterally administered CT, directly or indirectly, can inhibit a cell population in spleen which normally exerts an antigen-specific suppressive regulatory influence on the development of GVHR. | [
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PMID:26640 | [Acute mucocutaneous lymph node syndrome (Kawasaki-syndrome) with fatal periarteritis nodosa of the coronary arteries. Another case from Europe]. | Case report of a 1 8/12-year-old boy suffering from an acute febrile disease with phlegmonous cervical lymphadenopathy, skin rash and other septic symptoms. Bacterial cultures were sterile, and antibiotics had no effect, whereas prednisone made an immediate improvement. On the 23rd day of illness the patient died unexpectedly. Autopsy revealed a ruptured coronary aneurysm due to periarteritis nodosa. This curious disease of yet unknown cause is quite frequent in Japan, but very rare in Europe. | [Acute mucocutaneous lymph node syndrome (Kawasaki-syndrome) with fatal periarteritis nodosa of the coronary arteries. Another case from Europe]. Case report of a 1 8/12-year-old boy suffering from an acute febrile disease with phlegmonous cervical lymphadenopathy, skin rash and other septic symptoms. Bacterial cultures were sterile, and antibiotics had no effect, whereas prednisone made an immediate improvement. On the 23rd day of illness the patient died unexpectedly. Autopsy revealed a ruptured coronary aneurysm due to periarteritis nodosa. This curious disease of yet unknown cause is quite frequent in Japan, but very rare in Europe. | [
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PMID:26641 | Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature. | The histochemical activities of succinic dehydrogenase (SDH) and Ca++-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. 'Red' and 'white' muscle fibres, as distinguished by SDH, showed high and low Ca++-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K2-EDTA solution. The ultrastructural investigation of Ca++-ATPase reaction at pH 7.4 by the Ca++-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and 'Z' bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb++ precipitation technique demonstrated Mg++-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail 'red' muscle fibres are possible 'slow,' and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, 'red' muscle fibres of the anuran tai- musculature are not equivalent to 'Type I' fibres of higher chordates. | Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature. The histochemical activities of succinic dehydrogenase (SDH) and Ca++-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. 'Red' and 'white' muscle fibres, as distinguished by SDH, showed high and low Ca++-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K2-EDTA solution. The ultrastructural investigation of Ca++-ATPase reaction at pH 7.4 by the Ca++-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and 'Z' bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb++ precipitation technique demonstrated Mg++-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail 'red' muscle fibres are possible 'slow,' and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, 'red' muscle fibres of the anuran tai- musculature are not equivalent to 'Type I' fibres of higher chordates. | [
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PMID:26650 | Microbial metabolism of anthracycline antibiotics daunomycin and adriamycin. | It has been shown that the antitumor antibiotics daunomycin (1) and adriamycin (4) are metabolized by microorganisms in a fashion similar to their metabolism by mammalian cells. Both the fungus Mucor spinosus and its cell-free extracts reduce the 13-keto group of daunomycin to give daunomycinol (2) by a TPNH-dependent process. Cell-free extracts of Streptomyces steffisburgensis convert adriamycin and daunomycinol to their 7-deoxyaglycones (5) and (3) by DPNH-linked reductive glycosidic cleavage. Cell-free extracts of the latter organism convert 7-deoxyadriamycinone (5) to 7-deoxyadriamycinol aglycone (6) by TPNH-linked 13-keto reduction. | Microbial metabolism of anthracycline antibiotics daunomycin and adriamycin. It has been shown that the antitumor antibiotics daunomycin (1) and adriamycin (4) are metabolized by microorganisms in a fashion similar to their metabolism by mammalian cells. Both the fungus Mucor spinosus and its cell-free extracts reduce the 13-keto group of daunomycin to give daunomycinol (2) by a TPNH-dependent process. Cell-free extracts of Streptomyces steffisburgensis convert adriamycin and daunomycinol to their 7-deoxyaglycones (5) and (3) by DPNH-linked reductive glycosidic cleavage. Cell-free extracts of the latter organism convert 7-deoxyadriamycinone (5) to 7-deoxyadriamycinol aglycone (6) by TPNH-linked 13-keto reduction. | [
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PMID:26651 | Semiautomated turbidimetric microbiological assay for cefazaflur. | A quantitative semi-automated turbidimetric bioassay for cefazaflur, using Streptococcus faecium as the indicator, is described. Assays were run at pH 6.5 approximately 7 for 3.75 hours at 37 degrees C using 2 approximately 12 microgram cefazaflur per ml assay broth for standards. The dose response line was plotted point to point using the natural log of the absorbance vs natural log of the concentration. This assay is both accurate and precise and is more rapid than traditional plate assays for antibiotics. | Semiautomated turbidimetric microbiological assay for cefazaflur. A quantitative semi-automated turbidimetric bioassay for cefazaflur, using Streptococcus faecium as the indicator, is described. Assays were run at pH 6.5 approximately 7 for 3.75 hours at 37 degrees C using 2 approximately 12 microgram cefazaflur per ml assay broth for standards. The dose response line was plotted point to point using the natural log of the absorbance vs natural log of the concentration. This assay is both accurate and precise and is more rapid than traditional plate assays for antibiotics. | [
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PMID:26655 | Salmonella typhimurium peptidase active on carnosine. | Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium. | Salmonella typhimurium peptidase active on carnosine. Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium. | [
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PMID:26656 | Aspartokinase of Streptococcus mutans: purification, properties, and regulation. | Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine. | Aspartokinase of Streptococcus mutans: purification, properties, and regulation. Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine. | [
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PMID:26657 | Carboxypeptidase displaying differential velocity in hydrolysis of methotrexate, 5-methyltetrahydrofolic acid, and leucovorin. | An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extraced from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate. | Carboxypeptidase displaying differential velocity in hydrolysis of methotrexate, 5-methyltetrahydrofolic acid, and leucovorin. An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extraced from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate. | [
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PMID:26658 | Glutamate-induced uptake of proline by Streptomyces antibioticus. | Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized. | Glutamate-induced uptake of proline by Streptomyces antibioticus. Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized. | [
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PMID:26659 | Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase. | The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA. | Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase. The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA. | [
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PMID:26660 | Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase. | A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase. | Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase. A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase. | [
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PMID:26661 | Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. | The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism. | Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. The nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) of Chlorella sorokiniana was purified 260-fold to electrophoretic homogeneity in six steps. Depending on the techniques used, the native enzyme appeared to have a molecular weight of 290,000 or 410,000 and to be composed of five to seven identical subunits with a molecular weight of 58,000. The amino acid composition of this enzyme was shown to differ considerably from that of the NAD-GDH in this organism. The NH2-terminal amino acid was unavailable to dansylation. All six cysteines in the native enzyme were in the free sulfhydryl form. The pH optima for the aminating and deaminating reactions were 7.2 and 9.2, respectively. The Km values for NH4+, alpha-ketoglutarate, NADPH, L-glutamate, and NADP+ were 68, 12, 0.13, and 0.038 mM, respectively. At low substrate concentrations, no cooperativity was seen; however, severe inhibition of enzyme activity was observed at high alpha-ketoglutarate concentrations. Nucleotides did not affect enzyme activity. Antiserum produced in rabbits to the subunits of the enzyme yielded a single precipitin band with the purified enzyme in Ouchterlony double-diffusion analysis. Immunoelectrophoresis was used to confirm the purity of the enzyme and also to quantify the amount of enzyme antigen. These studies indicate that the NADPH-GDH and NAD-GDH isozymes are distinct molecular species in this organism. | [
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PMID:26662 | Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella. | When synchronous cells of the eucaryotic microorganism Chlorella sorokiniana growing in nitrate medium were challenged to synthesize an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) at frequent intervals during the cell cycle the initial rate of induction (i.e., enzyme potential) of this enzyme increased in an approximately linear manner until the period of DNA replication (i.e., S phase). During the S phase, NADP-GDH potential exhibited a positive rate change proportional to the step increase in DNA level. The timing of this rate change was insensitive to large changes in cellular growth rate. This rate change could be blocked within the first cell cycle by specific inhibition of DNA replication with 2'-deoxyadenosine. The approximately linear increase in NADP-GDH potential and also of total cellular protein observed before and after the S phase is proposed to be a result of the increasing photosynthetic capacity of the cell during the cell cycle. | Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella. When synchronous cells of the eucaryotic microorganism Chlorella sorokiniana growing in nitrate medium were challenged to synthesize an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) at frequent intervals during the cell cycle the initial rate of induction (i.e., enzyme potential) of this enzyme increased in an approximately linear manner until the period of DNA replication (i.e., S phase). During the S phase, NADP-GDH potential exhibited a positive rate change proportional to the step increase in DNA level. The timing of this rate change was insensitive to large changes in cellular growth rate. This rate change could be blocked within the first cell cycle by specific inhibition of DNA replication with 2'-deoxyadenosine. The approximately linear increase in NADP-GDH potential and also of total cellular protein observed before and after the S phase is proposed to be a result of the increasing photosynthetic capacity of the cell during the cell cycle. | [
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PMID:26663 | Mutations that alter the covalent modification of glutamine synthetase in Salmonella typhimurium. | glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction. | Mutations that alter the covalent modification of glutamine synthetase in Salmonella typhimurium. glnD and glnE mutant strains of Salmonella typhimurium lack three of the four activities required for reversible covalent modification of glutamine synthetase (GS; EC 6.3.1.2). The glnD strains, which are unable to deadenylylate GS and therefore accumulate the adenylylated or less active form of the enzyme, were isolated as glutamine bradytrophs. They lack the activity of PIIA uridylyl-transferase, one of the proteins required for deadenylylation of GS; in addition, they lack PIID uridylyl-removing activity. Mutations in glnD are suppressed by second-site mutations in glnE that eliminate the activity of GS adenylyltransferase (EC 2.7.7.42) and thus prevent adenylylation of GS. The glnD and glnE strains have one-third to one-half as much total GS as the wild-type strain when they are grown in a medium containing a high concentration of NH4+. The wild-type strain derepresses synthesis of GS fourfold in response to nitrogen limitation; glnD and glnE strains derepress synthesis of the enzyme fourfold and sevenfold, respectively. Thus, mutations that alter covalent modification of GS in Salmonella do not significantly affect derepression of its synthesis. The glnD gene lies at 7 min on the Salmonella chromosome and is 50% linked to pyrH by P22-mediated transduction. | [
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PMID:26664 | Genetics and physiology of Neurospora crassa glutamine auxotrophs. | This work reports on the isolation and characterization of two glutamine auxotrophs in Neurospora crassa. The mutations responsible for the glutamine-requiring phenotype were very closely linked, and one of them proved to be recessive to wild type. The mutations impaired the conversion of glutamic acid to glutamine and resulted in changes of both the activity and oligomeric structure of the enzyme glutamine synthetase. | Genetics and physiology of Neurospora crassa glutamine auxotrophs. This work reports on the isolation and characterization of two glutamine auxotrophs in Neurospora crassa. The mutations responsible for the glutamine-requiring phenotype were very closely linked, and one of them proved to be recessive to wild type. The mutations impaired the conversion of glutamic acid to glutamine and resulted in changes of both the activity and oligomeric structure of the enzyme glutamine synthetase. | [
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PMID:26665 | Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate. | Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum oxidation rates at pH 3.0, but produced substrate inhibition at other pH values. Tetrathionate-grown cell suspensions also oxidized thiosulfate at pH 2.0 to 4.0. Apparent Km values (1.2 to 25 mM) were of the same order as for tetrathionate, but kinetics were complex. Continuous culture on growth-limiting tetrathionate at pH 2.5, followed by continuous culture on growth-limiting thiosulfate at pH 2.5, indicated true growth yield values (grams [dry weight] per gram-molecule of substrate) of 12.2 and 7.5, and maintenance coefficient values (millimoles of substrate per gram [dry weight) of organisms per hour) of 1.01 and 0.97 for tetrathionate and thiosulfate, respectively. Yield was increased on both media at low dilution rates by increase in CO2 supply. The apparent maintenance coefficient was lowered without affecting YG, suggesting better energy coupling in CO2-rich environments. Prolonged continuous cultivation on tetrathionate or thiosulfate did not affect the ability of the organism to grow subsequently in ferrous iron medium. | Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate. Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum oxidation rates at pH 3.0, but produced substrate inhibition at other pH values. Tetrathionate-grown cell suspensions also oxidized thiosulfate at pH 2.0 to 4.0. Apparent Km values (1.2 to 25 mM) were of the same order as for tetrathionate, but kinetics were complex. Continuous culture on growth-limiting tetrathionate at pH 2.5, followed by continuous culture on growth-limiting thiosulfate at pH 2.5, indicated true growth yield values (grams [dry weight] per gram-molecule of substrate) of 12.2 and 7.5, and maintenance coefficient values (millimoles of substrate per gram [dry weight) of organisms per hour) of 1.01 and 0.97 for tetrathionate and thiosulfate, respectively. Yield was increased on both media at low dilution rates by increase in CO2 supply. The apparent maintenance coefficient was lowered without affecting YG, suggesting better energy coupling in CO2-rich environments. Prolonged continuous cultivation on tetrathionate or thiosulfate did not affect the ability of the organism to grow subsequently in ferrous iron medium. | [
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PMID:26666 | Sodium ion-proton antiport in a marine bacterium. | Alteromonas haloplanktis ejected protons in response to a brief respiratory pulse; the rate of decay of the resulting pH change was accelerated when Na+ was present in the suspension medium. The addition of an anaerobic NaCl solution to an essentially Na+-free anaerobic bacterial suspension induced the acidification of the suspension medium. These results and others discussed provide substantial evidence for the existence of an Na+-H+ antiporter in this organism. | Sodium ion-proton antiport in a marine bacterium. Alteromonas haloplanktis ejected protons in response to a brief respiratory pulse; the rate of decay of the resulting pH change was accelerated when Na+ was present in the suspension medium. The addition of an anaerobic NaCl solution to an essentially Na+-free anaerobic bacterial suspension induced the acidification of the suspension medium. These results and others discussed provide substantial evidence for the existence of an Na+-H+ antiporter in this organism. | [
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PMID:26668 | Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma). | The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure. | Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma). The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure. | [
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PMID:26667 | Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida. | The primary amine dehydrogenase of Pseudomonas putida NP was purified to homogeneity as judged by polyacrylamide gel electrophoresis. Cytochrome c or an artificial electron acceptor was required for amine dehydrogenase activity. The enzyme was nonspecific, readily oxidizing primary monoamines, benzylamine, and tyramine; little or no measurable activity was detected with isoamines, L-ornithine, L-lysine, and certain diamines or polyamines. The pH optima for n-butylamine, benzylamine, and n-propylamine were 7.0, 6.5, and 7.0, respectively. The molecular weight of the enzyme was 112,000 as determined by gel filtration and 95,300 as analyzed by sedimentation equilibrium. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested that the enzyme was composed of two nonidentical subunits with molecular weights of 58,000 and 42,000. The absorption spectrum of the purified enzyme was indicative of a hemoprotein, exhibiting absorption maxima at 277, 355, and 408 nm. Reduction with sodium dithionite or amine substrates resulted in absorption maxima at 523 and 552 nm and a shift in the Soret peak to 416 nm. These results suggested that the enzyme is a hemoprotein of the type c cytochrome. There was no evidence that flavins were present. | Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida. The primary amine dehydrogenase of Pseudomonas putida NP was purified to homogeneity as judged by polyacrylamide gel electrophoresis. Cytochrome c or an artificial electron acceptor was required for amine dehydrogenase activity. The enzyme was nonspecific, readily oxidizing primary monoamines, benzylamine, and tyramine; little or no measurable activity was detected with isoamines, L-ornithine, L-lysine, and certain diamines or polyamines. The pH optima for n-butylamine, benzylamine, and n-propylamine were 7.0, 6.5, and 7.0, respectively. The molecular weight of the enzyme was 112,000 as determined by gel filtration and 95,300 as analyzed by sedimentation equilibrium. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested that the enzyme was composed of two nonidentical subunits with molecular weights of 58,000 and 42,000. The absorption spectrum of the purified enzyme was indicative of a hemoprotein, exhibiting absorption maxima at 277, 355, and 408 nm. Reduction with sodium dithionite or amine substrates resulted in absorption maxima at 523 and 552 nm and a shift in the Soret peak to 416 nm. These results suggested that the enzyme is a hemoprotein of the type c cytochrome. There was no evidence that flavins were present. | [
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PMID:26669 | Organic fluorides: implications for psychiatry. | The widespread use of synthetic organic fluorides has recently received attention as a potential health hazard. There are a number of organic fluorides which have become important considerations in psychiatry. Therapeutically the organic fluorides include the neuroleptics trifluoperazine, fluphenazine, triflupromazine, and haloperidol, the benzodiazepine flurazepam and the polyfluorinated inhalant convulsant indoklon. On the negative side, deliberate inhalation of fluorocarbon aerosol propellants has become a modern form of drug abuse among the young. A review is presented on the biochemistry and toxicology of organic fluorides with special emphasis on implications to the field of psychiatry. | Organic fluorides: implications for psychiatry. The widespread use of synthetic organic fluorides has recently received attention as a potential health hazard. There are a number of organic fluorides which have become important considerations in psychiatry. Therapeutically the organic fluorides include the neuroleptics trifluoperazine, fluphenazine, triflupromazine, and haloperidol, the benzodiazepine flurazepam and the polyfluorinated inhalant convulsant indoklon. On the negative side, deliberate inhalation of fluorocarbon aerosol propellants has become a modern form of drug abuse among the young. A review is presented on the biochemistry and toxicology of organic fluorides with special emphasis on implications to the field of psychiatry. | [
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PMID:26670 | Enzymatic oxidation of disulfides and thiolsulfinates by both rabbit liver microsomes and a reconstituted system with purified cytochrome P-450. | Both rabbit liver microsomes and reconstituted system with purified cytochrome P-450 and cofactors enzymatically oxidized o-dithiane (1, 2-dithiane), 3-methyl-o-dithiane, thiane and 2-methylthiane to the corresponding mono-oxygenated products; sulfides or disulfides were oxidized to the corresponding sulfoxides or thiosulfinates, while thiosulfinate was oxidized to thiolsulfonate. The reconstituted systems required oxygen and NADPH and were not affected by the catalase which decomposes H2O2, or by 1,4-diazabicyclo-[2,2,2]octane (DABCO), which is a good quencher of singlet oxygen. The differences in the binding of substrates such as sulfides and disulfides with the enzyme system are discussed in connection with differences in the spectra of the substrates in the reconstituted system with pure cytochrome P-450. A correlation was found between the rates of oxidation of the substrates and the rates of oxidation of NADPH. | Enzymatic oxidation of disulfides and thiolsulfinates by both rabbit liver microsomes and a reconstituted system with purified cytochrome P-450. Both rabbit liver microsomes and reconstituted system with purified cytochrome P-450 and cofactors enzymatically oxidized o-dithiane (1, 2-dithiane), 3-methyl-o-dithiane, thiane and 2-methylthiane to the corresponding mono-oxygenated products; sulfides or disulfides were oxidized to the corresponding sulfoxides or thiosulfinates, while thiosulfinate was oxidized to thiolsulfonate. The reconstituted systems required oxygen and NADPH and were not affected by the catalase which decomposes H2O2, or by 1,4-diazabicyclo-[2,2,2]octane (DABCO), which is a good quencher of singlet oxygen. The differences in the binding of substrates such as sulfides and disulfides with the enzyme system are discussed in connection with differences in the spectra of the substrates in the reconstituted system with pure cytochrome P-450. A correlation was found between the rates of oxidation of the substrates and the rates of oxidation of NADPH. | [
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PMID:26673 | New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase. | L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate. | New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase. L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate. | [
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PMID:26674 | Acceptor specificity of ATP:nucleoside-5'-phosphate pyrophosphotransferase from Streptomyces adephospholyticus. Synthesis of the 3'-pyrophosphates of pyrimidine nucleotides, some oligoribonucleotides, 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am. | ATP: nucleoside-5'-phosphate pyrophosphotransferase [EC 2.7.6.4] of Streptomyces adephospholyticus synthesizes not only 3'-pyrophosphates of 5'-purine ribomononucleotides but also those of pyrimidine mononucleotides, some short oligonucleotides, a variety of 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am. | Acceptor specificity of ATP:nucleoside-5'-phosphate pyrophosphotransferase from Streptomyces adephospholyticus. Synthesis of the 3'-pyrophosphates of pyrimidine nucleotides, some oligoribonucleotides, 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am. ATP: nucleoside-5'-phosphate pyrophosphotransferase [EC 2.7.6.4] of Streptomyces adephospholyticus synthesizes not only 3'-pyrophosphates of 5'-purine ribomononucleotides but also those of pyrimidine mononucleotides, some short oligonucleotides, a variety of 5'-diphosphonucleosidic coenzymes and mG-5'-ppp-5'-Am. | [
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PMID:26675 | Purification and characterization of alkaline phosphatase in cultured rat liver cells. | Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4. | Purification and characterization of alkaline phosphatase in cultured rat liver cells. Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4. | [
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PMID:26676 | Purification of lysophospholipase of Vibrio parahaemolyticus and its properties. | Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water. | Purification of lysophospholipase of Vibrio parahaemolyticus and its properties. Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water. | [
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PMID:26677 | Studies on the formation of gamma-aminobutyric acid from putrescine in rat organs and purification of its synthetic enzyme from rat intestine. | The formation of gamma-aminobutyric acid (GABA) from putrescine was examined in organs of rats using radioactive putrescine. Radioactive GABA was detected in all the organs of a rat injected intraperitoneally with radioactive putrescine, and the highest radioactivity of GABA was observed in the small intestine. The enzyme involved in this formation was purified from small intestine and identified as a diamine oxidase, histaminase, from the properties of the enzyme. Activity of the enzyme was found in all the organs of rat, and the highest activity was observed in the small intestine. | Studies on the formation of gamma-aminobutyric acid from putrescine in rat organs and purification of its synthetic enzyme from rat intestine. The formation of gamma-aminobutyric acid (GABA) from putrescine was examined in organs of rats using radioactive putrescine. Radioactive GABA was detected in all the organs of a rat injected intraperitoneally with radioactive putrescine, and the highest radioactivity of GABA was observed in the small intestine. The enzyme involved in this formation was purified from small intestine and identified as a diamine oxidase, histaminase, from the properties of the enzyme. Activity of the enzyme was found in all the organs of rat, and the highest activity was observed in the small intestine. | [
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PMID:26678 | Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma. | Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far. | Purification and characterization of alkaline phosphatase from plasma membranes of rat ascites hepatoma. Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotien, was homogeneous in polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of beta-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5--10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km-0.3 mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; beta-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36% and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55 degrees C for 30 min, or exposed either to 10 mM beta-mercaptoethanol for 10 min to 3 M urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20% whereas Zn2+ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far. | [
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PMID:26679 | Monomer-dimer equilibria of a Bence Jones protein and its variable fragment. | The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains. | Monomer-dimer equilibria of a Bence Jones protein and its variable fragment. The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains. | [
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PMID:26680 | A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin. | Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath. | A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin. Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath. | [
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PMID:26682 | 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. | The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism. | 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism. | [
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PMID:26685 | The protonmotive force and beta-galactoside transport in Bacillus acidocaldarius. | The acidophilic and thermophilic bacterium, Bacillus acidocaldarius maintains a cytoplasmic pH between 5.85 and 6.31 over a range of external pH from 2.0 to 4.5. Consistently, the pH optimum of beta-galactosidase, as assayed in cell extracts, is between pH 6.0 and 6.5. An electrical potential (delta-psi), interior positive, is also maintained across the membrane. A delta-psi of approximately 34 mV was calculated from determinations of thiocyanate uptake by cells at pH 3.5. Addition of the proton conductor carbonyl cyanide m-chlorophenylhydrazone increased the delta-psi. Treatment of cells with valinomycin (in the absence of external potassium ions) or high concentrations of thiocyanate, to abolish the delta psi, resulted in collapse of the transmembrane proton gradient (delta pH). Active transport of methylthio-beta, D-galactoside occurred optimally at pH 3.5. Transport of the galactoside was inhibited by various compounds which could dissipate the transmembrane delta pH and by respiratory inhibitors. A decrease in the delta pH and an increase in the delta psi occurred upon addition of methylthio-beta, D-galactoside to cells of B. acidocaldarius. Thus the transport of this solute appears to involve an electrogenic symport with protons. The transport system is most active at 50 degrees C and shows little activity at 25 degrees C, although the delta pH is the same at the two temperatures. Gramicidin inhibits methylthio-beta, D-galactoside transport equally effectively at 50 degrees C and 25 degrees C, while nigericin inhibits only after a lag at 25 degrees C. | The protonmotive force and beta-galactoside transport in Bacillus acidocaldarius. The acidophilic and thermophilic bacterium, Bacillus acidocaldarius maintains a cytoplasmic pH between 5.85 and 6.31 over a range of external pH from 2.0 to 4.5. Consistently, the pH optimum of beta-galactosidase, as assayed in cell extracts, is between pH 6.0 and 6.5. An electrical potential (delta-psi), interior positive, is also maintained across the membrane. A delta-psi of approximately 34 mV was calculated from determinations of thiocyanate uptake by cells at pH 3.5. Addition of the proton conductor carbonyl cyanide m-chlorophenylhydrazone increased the delta-psi. Treatment of cells with valinomycin (in the absence of external potassium ions) or high concentrations of thiocyanate, to abolish the delta psi, resulted in collapse of the transmembrane proton gradient (delta pH). Active transport of methylthio-beta, D-galactoside occurred optimally at pH 3.5. Transport of the galactoside was inhibited by various compounds which could dissipate the transmembrane delta pH and by respiratory inhibitors. A decrease in the delta pH and an increase in the delta psi occurred upon addition of methylthio-beta, D-galactoside to cells of B. acidocaldarius. Thus the transport of this solute appears to involve an electrogenic symport with protons. The transport system is most active at 50 degrees C and shows little activity at 25 degrees C, although the delta pH is the same at the two temperatures. Gramicidin inhibits methylthio-beta, D-galactoside transport equally effectively at 50 degrees C and 25 degrees C, while nigericin inhibits only after a lag at 25 degrees C. | [
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PMID:26686 | The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli. | Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit. | The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli. Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit. | [
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PMID:26687 | Indoleamine 2,3-dioxygenase. Purification and some properties. | Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity. | Indoleamine 2,3-dioxygenase. Purification and some properties. Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity. | [
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PMID:26689 | Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure. | The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles. | Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure. The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles. | [
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PMID:26690 | Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes. | The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs. | Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes. The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs. | [
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PMID:26691 | A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography. | A simplified high-pressure liquid chromatograhic method for determination of furosemide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C15 hydrocarbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 microgram per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown. | A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography. A simplified high-pressure liquid chromatograhic method for determination of furosemide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C15 hydrocarbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 microgram per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown. | [
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PMID:26692 | High-voltage paper electrophoretic assay for guanyl cyclase. | A simple, sensitive and rapid technique is described, permitting separation of cGMP from GMP, GDP and GTP by the use of unidirectional high-voltage paper electrophoresis. The recovery of labeled cGMP in the assay of guanyl cyclase, by this procedure is 85-90%; the blank values (no enzyme) are negligible. | High-voltage paper electrophoretic assay for guanyl cyclase. A simple, sensitive and rapid technique is described, permitting separation of cGMP from GMP, GDP and GTP by the use of unidirectional high-voltage paper electrophoresis. The recovery of labeled cGMP in the assay of guanyl cyclase, by this procedure is 85-90%; the blank values (no enzyme) are negligible. | [
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PMID:26693 | Gentamicin-blood agar for isolation of Streptococcus pneumoniae from respiratory secretions. | Previous studies have suggested that the yield of Streptococcus pneumoniae from respiratory secretions can be increased by using a 5% sheep blood agar plate supplemented with 5 microgram of gentamicin (GBA) per ml. We report our experience with 245 lower respiratory specimens in which this method was compared with 5% sheep blood agar (SBA) alone. Of 35 specimens with growth of S. pneumoniae on either plate, 21 were detected exclusively on SBA, whereas only 3 were detected on GBA alone (P less than 0.01). By subculturing representative alpha-hemolytic colonies from the final 169 specimens, the yield of S. pneumoniae was increased by 27% compared with the number of identifications that could be made directly from the primary culture. Minimal inhibitory concentrations of gentamicin for the last 25 isolates were greater than or equal to 8 microgram/ml. Our results do not substantiate the previous observations that S. pneumoniae from respiratory secretions gives an increased yield in cultures on GBA. | Gentamicin-blood agar for isolation of Streptococcus pneumoniae from respiratory secretions. Previous studies have suggested that the yield of Streptococcus pneumoniae from respiratory secretions can be increased by using a 5% sheep blood agar plate supplemented with 5 microgram of gentamicin (GBA) per ml. We report our experience with 245 lower respiratory specimens in which this method was compared with 5% sheep blood agar (SBA) alone. Of 35 specimens with growth of S. pneumoniae on either plate, 21 were detected exclusively on SBA, whereas only 3 were detected on GBA alone (P less than 0.01). By subculturing representative alpha-hemolytic colonies from the final 169 specimens, the yield of S. pneumoniae was increased by 27% compared with the number of identifications that could be made directly from the primary culture. Minimal inhibitory concentrations of gentamicin for the last 25 isolates were greater than or equal to 8 microgram/ml. Our results do not substantiate the previous observations that S. pneumoniae from respiratory secretions gives an increased yield in cultures on GBA. | [
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PMID:26694 | Diagnosis of pneumococcal pneumonia by antigen detection in sputum. | Pneumococcal polysaccharide was detected by counterimmunoelectrophoresis in the sputum of 20 of 26 (77%) adults with community-acquired pneumonia and a positive sputum culture for Streptococcus pneumoniae. The test was negative in 29 pneumonia patients with negative sputum culture for S. pneumoniae. Pneumococcal antigen was also detected in the sputum of six of nine adults with chronic bronchitis and a positive sputum culture, but was not detected in expectorated respiratory secretions of 22 pneumococcal carriers with colds. Pneumococcal antigen could also be detected in sputum by immunodiffusion; antigen titers varied from 1:2 to 1:256. These results strongly suggest that the detection of pneumococcal antigen in respiratory tract secretions indicates infection caused by S. pneumoniae. | Diagnosis of pneumococcal pneumonia by antigen detection in sputum. Pneumococcal polysaccharide was detected by counterimmunoelectrophoresis in the sputum of 20 of 26 (77%) adults with community-acquired pneumonia and a positive sputum culture for Streptococcus pneumoniae. The test was negative in 29 pneumonia patients with negative sputum culture for S. pneumoniae. Pneumococcal antigen was also detected in the sputum of six of nine adults with chronic bronchitis and a positive sputum culture, but was not detected in expectorated respiratory secretions of 22 pneumococcal carriers with colds. Pneumococcal antigen could also be detected in sputum by immunodiffusion; antigen titers varied from 1:2 to 1:256. These results strongly suggest that the detection of pneumococcal antigen in respiratory tract secretions indicates infection caused by S. pneumoniae. | [
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PMID:26695 | Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system. | Stimulation of guinea pig granolocytes by digitonin results in superoxide (O-2) generation. A continuous assay shows that there is a lag between the addition of digitonin and the onset of O-2 production. The rate of activation of the O-2 generating system is dependent upon the concentration of digitonin and the temperature. The final linear rate of O-2 production is affected by the concentration of digitonin, temperature, pH, and the presence of exogenous reduced pyridine nucleotides. Thus, factors which alter either the activation process or the activity of the O-2 generating system can affect O-2 production by stimulated granolocytes. | Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system. Stimulation of guinea pig granolocytes by digitonin results in superoxide (O-2) generation. A continuous assay shows that there is a lag between the addition of digitonin and the onset of O-2 production. The rate of activation of the O-2 generating system is dependent upon the concentration of digitonin and the temperature. The final linear rate of O-2 production is affected by the concentration of digitonin, temperature, pH, and the presence of exogenous reduced pyridine nucleotides. Thus, factors which alter either the activation process or the activity of the O-2 generating system can affect O-2 production by stimulated granolocytes. | [
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PMID:26696 | Inhibition of the bicarbonate exit step in urinary acidification by a disulfonic stilbene. | Acidification of the luminal solution by the isolated turtle bladder involves H(+) secretion by a pump at the luminal membrane. The OH(-) dissociated in this process reacts with CO(2) and forms HCO(3) (-) which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO(3) (-) was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H(+) secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H(+) secretion by serosal SITS, overall cell pH, measured by the 5, 5-dimethyl-2, 3-oxazolidinedione method, increased from 7.48+/-0.03 to 7.61+/-0.02. This increase of 0.13+/-0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88+/-0.06 to 2.09+/-0.11 pH U. In the absence of evidence for increased H(+) permeability or a change in the force of the H(+) pump, the gradient decrement of 0.79+/-0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump. The increased electrochemical gradient across the active transport pathway is the primary factor in the inhibition of urinary acidification. | Inhibition of the bicarbonate exit step in urinary acidification by a disulfonic stilbene. Acidification of the luminal solution by the isolated turtle bladder involves H(+) secretion by a pump at the luminal membrane. The OH(-) dissociated in this process reacts with CO(2) and forms HCO(3) (-) which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO(3) (-) was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H(+) secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H(+) secretion by serosal SITS, overall cell pH, measured by the 5, 5-dimethyl-2, 3-oxazolidinedione method, increased from 7.48+/-0.03 to 7.61+/-0.02. This increase of 0.13+/-0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88+/-0.06 to 2.09+/-0.11 pH U. In the absence of evidence for increased H(+) permeability or a change in the force of the H(+) pump, the gradient decrement of 0.79+/-0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump. The increased electrochemical gradient across the active transport pathway is the primary factor in the inhibition of urinary acidification. | [
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PMID:26697 | An androgen binding protein in the testicular cytosol of human testis. Comparison with human plasma testosterone-estrogen binding globulin. | After removal of plasma contamination, an androgen binding protein (hABP) was detected in a 105,000-g supernate of human testicular homogenate. The physicochemical properties of hABP have been compared with a similar androgen binding protein in human plasma, testosterone-estrogen binding globulin (TeBG). hABP had high affinity (K(d) = 7.8 nM) and low capacity (0.27 pmol/mg protein) for 5alpha-dihydrotestosterone (DHT). Binding affinity of human TeBG for DHT was greater (K(d) = 0.66 nM, binding capacity 0.68 pmol/mg protein). On the basis of sedimentation rates and Einstein Stokes radii of hABP and TeBG, the mol wt of the two proteins were similar in the range of 87,000-92,000. The ligand specificities of hABP and TeBG were the same. The binding of [(3)H]DHT to hABP and TeBG were reversible processes at 0 degrees C. The half-lives for the dissociation of [(3)H]DHT from hABP and TeBG were 100-120 min and 67-70 min, respectively. Heat sensitivity of hABP and TeBG were similar. hABP had a sharp pH binding curve with an optimum at 8, whereas TeBG had a stable pH optimum between 6.5 and 9. hABP and TeBG were eluted from an ion exchange column at 100 mM and 80 mM sodium chloride concentrations, respectively. Concanavalin A and ricin Sepharose affinity chromatography showed that TeBG is bound to the columns nearly quantitatively, whereas hABP is bound to the columns only partially. Differences between hABP and TeBG, plus the reduction of plasma contamination as marked by albumin, suggest that the human mature testis contains an androgen binding protein separate from that circulating in plasma. | An androgen binding protein in the testicular cytosol of human testis. Comparison with human plasma testosterone-estrogen binding globulin. After removal of plasma contamination, an androgen binding protein (hABP) was detected in a 105,000-g supernate of human testicular homogenate. The physicochemical properties of hABP have been compared with a similar androgen binding protein in human plasma, testosterone-estrogen binding globulin (TeBG). hABP had high affinity (K(d) = 7.8 nM) and low capacity (0.27 pmol/mg protein) for 5alpha-dihydrotestosterone (DHT). Binding affinity of human TeBG for DHT was greater (K(d) = 0.66 nM, binding capacity 0.68 pmol/mg protein). On the basis of sedimentation rates and Einstein Stokes radii of hABP and TeBG, the mol wt of the two proteins were similar in the range of 87,000-92,000. The ligand specificities of hABP and TeBG were the same. The binding of [(3)H]DHT to hABP and TeBG were reversible processes at 0 degrees C. The half-lives for the dissociation of [(3)H]DHT from hABP and TeBG were 100-120 min and 67-70 min, respectively. Heat sensitivity of hABP and TeBG were similar. hABP had a sharp pH binding curve with an optimum at 8, whereas TeBG had a stable pH optimum between 6.5 and 9. hABP and TeBG were eluted from an ion exchange column at 100 mM and 80 mM sodium chloride concentrations, respectively. Concanavalin A and ricin Sepharose affinity chromatography showed that TeBG is bound to the columns nearly quantitatively, whereas hABP is bound to the columns only partially. Differences between hABP and TeBG, plus the reduction of plasma contamination as marked by albumin, suggest that the human mature testis contains an androgen binding protein separate from that circulating in plasma. | [
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PMID:26702 | Relationship between phosphorylation of tyrosine aminotransferase and regulation of its synthesis by cyclic AMP and hormones. | Tyrosine adminotransferase (EC 2.6.1.5) has been found to be phosphorylated in intact rat hepatoma cells in culture. Incorporation of [32p]i into the enzyme is rapid and is exclusively found as phosphoserine. Cycloheximide treatment reduced phosphorylation of the aminotransferase only slightly and in the presence of three different inducers of this enzyme, dexamethasone, insulin, and dibutyryl cyclic AMP, [32P]I incorporation was increased. It is concluded that [32p]i incorporation into this enzyme probably reflects turnover of phosphate groups associated with pre-existing enzyme molecules catalyzed by a cyclic AMP-independent protein kinase. | Relationship between phosphorylation of tyrosine aminotransferase and regulation of its synthesis by cyclic AMP and hormones. Tyrosine adminotransferase (EC 2.6.1.5) has been found to be phosphorylated in intact rat hepatoma cells in culture. Incorporation of [32p]i into the enzyme is rapid and is exclusively found as phosphoserine. Cycloheximide treatment reduced phosphorylation of the aminotransferase only slightly and in the presence of three different inducers of this enzyme, dexamethasone, insulin, and dibutyryl cyclic AMP, [32P]I incorporation was increased. It is concluded that [32p]i incorporation into this enzyme probably reflects turnover of phosphate groups associated with pre-existing enzyme molecules catalyzed by a cyclic AMP-independent protein kinase. | [
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PMID:26703 | The stimulation by catecholamines of guanylate cyclase activity in a cell-free system. | Cytosolic guanylate cylase activity in cell-free preparations of the rabbit renal cortex was increased 3- to 5-fold by catecholamines. The plasma membrane-bound enzyme was not activated, although hormone receptors were present. Stimulation was augmented by NaN3, which by itself had little effect on the soluble enzyme activity. With a partially purified enzyme, activity was enhanced by 0.1 muM 1-epinephrine and activated half-maximally by about 1 muM. In decreasing potency, epinephrine greater than isoproterenol greater than norepinephrine greater than dopamine greater than catechol. Phenylephrine and metanephrine did not stimulate. 1-Epinephrine-stimulation of the enzyme was reversed by dialysis and the deactivated enzyme was reactivatable by a second exposure to the catecholamine. Activation by catecholamines was not stereospecific. Epinephrine-stimulated guanylate cyclase activity in the crude cytosolic fraction was partially inhibited by alpha-adrenergic antagonists, but neither alpha- nor beta-blockers inhibited when the partially purified enzyme was used; thus, leaving open the question of a role for typical alpha- or beta-adrenergic mechanisms in this regulation of the soluble enzyme. Adrenochrome was the most potent activator of the partially purified guanylate cyclase, being approximately 10-times more effective than epinephrine. Epinephrine and adrenochrome activated in the presence of reducing agents, i.e., ascorbate, DTT and N2, although the enzyme in a more SH-reduced form and in an oxygen-deficient medium had a decreased sensitivity to both effectors. Epinephrine activated soluble guanylate cyclase in several tissues, including cerebrum, cerebellum, brain stem, lung, heart, liver, ductus deferens and colon. Although the precise mechanism by which low concentrations of catecholamines stimulated guanylate cyclase activity is unknown and the physiological significance of the activation remains to be established, these findings direct attention to an interesting interaction of catecholamines with the cytosolic enzyme system and stress the need for further studies. | The stimulation by catecholamines of guanylate cyclase activity in a cell-free system. Cytosolic guanylate cylase activity in cell-free preparations of the rabbit renal cortex was increased 3- to 5-fold by catecholamines. The plasma membrane-bound enzyme was not activated, although hormone receptors were present. Stimulation was augmented by NaN3, which by itself had little effect on the soluble enzyme activity. With a partially purified enzyme, activity was enhanced by 0.1 muM 1-epinephrine and activated half-maximally by about 1 muM. In decreasing potency, epinephrine greater than isoproterenol greater than norepinephrine greater than dopamine greater than catechol. Phenylephrine and metanephrine did not stimulate. 1-Epinephrine-stimulation of the enzyme was reversed by dialysis and the deactivated enzyme was reactivatable by a second exposure to the catecholamine. Activation by catecholamines was not stereospecific. Epinephrine-stimulated guanylate cyclase activity in the crude cytosolic fraction was partially inhibited by alpha-adrenergic antagonists, but neither alpha- nor beta-blockers inhibited when the partially purified enzyme was used; thus, leaving open the question of a role for typical alpha- or beta-adrenergic mechanisms in this regulation of the soluble enzyme. Adrenochrome was the most potent activator of the partially purified guanylate cyclase, being approximately 10-times more effective than epinephrine. Epinephrine and adrenochrome activated in the presence of reducing agents, i.e., ascorbate, DTT and N2, although the enzyme in a more SH-reduced form and in an oxygen-deficient medium had a decreased sensitivity to both effectors. Epinephrine activated soluble guanylate cyclase in several tissues, including cerebrum, cerebellum, brain stem, lung, heart, liver, ductus deferens and colon. Although the precise mechanism by which low concentrations of catecholamines stimulated guanylate cyclase activity is unknown and the physiological significance of the activation remains to be established, these findings direct attention to an interesting interaction of catecholamines with the cytosolic enzyme system and stress the need for further studies. | [
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PMID:26704 | gamma-Glutamyl transpeptidase in lactating mammary secretory tissue of cow and rat. | Lactating cow and rat tissues were examined for gamma-glutamyl transpeptidase. The specificity of the enzyme for various amino acid acceptors was determined for mammary tissue. The relative specific activity of gamma-glutamyl transpeptidase was high in cow mammary tissue and lower in rat mammary tissue in relation to the activity in kidney of the respective mammals. However, activity per gram wet weight of mammary tissues appeared equivalent. gamma-Glutamyl transpeptidase activity towards specific amino acids agreed with the percent extractions of these amino acids by the mammary gland of the lactating cow in vivo. We believe this is the first evidence of a possible system of amino acid transport by mammary tissues. | gamma-Glutamyl transpeptidase in lactating mammary secretory tissue of cow and rat. Lactating cow and rat tissues were examined for gamma-glutamyl transpeptidase. The specificity of the enzyme for various amino acid acceptors was determined for mammary tissue. The relative specific activity of gamma-glutamyl transpeptidase was high in cow mammary tissue and lower in rat mammary tissue in relation to the activity in kidney of the respective mammals. However, activity per gram wet weight of mammary tissues appeared equivalent. gamma-Glutamyl transpeptidase activity towards specific amino acids agreed with the percent extractions of these amino acids by the mammary gland of the lactating cow in vivo. We believe this is the first evidence of a possible system of amino acid transport by mammary tissues. | [
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PMID:26707 | Effect of endosulfan on drug metabolizing enzymes and lipid peroxidation in rat. | The influence of endosulfan, a chlorinated hydrocarbon insecticide, on rat hepatic drug metabolizing enzymes and lipid peroxidation has been explored in the present study. Total liver weight was significantly increased after endosulfan administration at both dose levels (2.5 and 5.0 mg/kg body weight) without affecting body weight or 9000 x g supernatant protein content. There was a marked increase in the activity of aminopyrine-N-demethylase, aniline hydroxylase, tyrosine amino-transferase and spontaneous lipid peroxidation. In all cases the increase was dose dependent. | Effect of endosulfan on drug metabolizing enzymes and lipid peroxidation in rat. The influence of endosulfan, a chlorinated hydrocarbon insecticide, on rat hepatic drug metabolizing enzymes and lipid peroxidation has been explored in the present study. Total liver weight was significantly increased after endosulfan administration at both dose levels (2.5 and 5.0 mg/kg body weight) without affecting body weight or 9000 x g supernatant protein content. There was a marked increase in the activity of aminopyrine-N-demethylase, aniline hydroxylase, tyrosine amino-transferase and spontaneous lipid peroxidation. In all cases the increase was dose dependent. | [
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PMID:26709 | Onchocerca sweetae (Nematoda: Filarioidea): notes on the intermediate host. | Microfilariae of Onchocerca sweetae are broadly distributed in the superficial layers of the dermis of the water buffalo (Bubalus bubalis). A total of 2855 insects representing 20 species were collected from O. sweetae-infected bait buffaloes. Only one species, Culicoides sp. "M", ingested microfilariae from buffalo skin. Larval development of O. sweetae was observed in the thorax of this species. Atotal of 829 insects, representing 7 species and including 749 parous Culicoides spp. were collected from light and Manitoba traps. Developing filarioid larvae were observed only in Culicoides sp. "M". It is concluded that Culicoides sp. "M" is a natural intermediate host of O. sweetae in the Northern Territory of Australia. | Onchocerca sweetae (Nematoda: Filarioidea): notes on the intermediate host. Microfilariae of Onchocerca sweetae are broadly distributed in the superficial layers of the dermis of the water buffalo (Bubalus bubalis). A total of 2855 insects representing 20 species were collected from O. sweetae-infected bait buffaloes. Only one species, Culicoides sp. "M", ingested microfilariae from buffalo skin. Larval development of O. sweetae was observed in the thorax of this species. Atotal of 829 insects, representing 7 species and including 749 parous Culicoides spp. were collected from light and Manitoba traps. Developing filarioid larvae were observed only in Culicoides sp. "M". It is concluded that Culicoides sp. "M" is a natural intermediate host of O. sweetae in the Northern Territory of Australia. | [
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PMID:26717 | Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts. | The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies. | Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts. The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies. | [
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PMID:26718 | Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man. | The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD. | Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man. The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD. | [
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PMID:26719 | Pharmacologic control of the hormonally modulated immune response. III. Prolongation of allogeneic skin graft rejection and prevention of runt disease by a combination of drugs acting on neuroendocrine functions. | A recently developed pharmacologic means for suppressing acquired immunity by drugs acting on neuroendocrine regulation has been applied to transplantation immune reactions. A number of drugs have been tested singly and in combination for their capacity to suppress the immune response of mice grafted with allogeneic skin. Another model involved newborn F1 hybrid recipients inoculated with spleen cells from donors of parental strains that had been made specifically "unresponsive" by drug and alloantigen treatment. These procedures led to the identification of a combination of four drugs that induced a remarkable delay in allograft rejection and a prolonged unresponsiveness to alloantigens. This combination of drugs also abrogated the graft-vs-host-runting syndrome in newborn hybrid recipients. | Pharmacologic control of the hormonally modulated immune response. III. Prolongation of allogeneic skin graft rejection and prevention of runt disease by a combination of drugs acting on neuroendocrine functions. A recently developed pharmacologic means for suppressing acquired immunity by drugs acting on neuroendocrine regulation has been applied to transplantation immune reactions. A number of drugs have been tested singly and in combination for their capacity to suppress the immune response of mice grafted with allogeneic skin. Another model involved newborn F1 hybrid recipients inoculated with spleen cells from donors of parental strains that had been made specifically "unresponsive" by drug and alloantigen treatment. These procedures led to the identification of a combination of four drugs that induced a remarkable delay in allograft rejection and a prolonged unresponsiveness to alloantigens. This combination of drugs also abrogated the graft-vs-host-runting syndrome in newborn hybrid recipients. | [
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PMID:26723 | Antibody response to capsular polysaccharide vaccine of Streptococcus pneumoniae in patients with nephrotic syndrome. | Twenty-three normal subjects and 19 patients with nephrotic syndrome were vaccinated with tridecavalent pneumococcal capsular polysaccharide vaccine of Streptococcus pneumoniae. The antibody response of the nephrotic patients to pneumococcal capsular antigens was equal to that of the control subjects. These findings indicate that patients with nephrotic syndrome, despite hypogammaglobulinemia, can mount an adequate antibody response to pneumococcal polysaccharides and that there is no evidence of suppressor thymus-derived (T) cells of dysfunctioning bone marrow-derived (B) cells in these patients. | Antibody response to capsular polysaccharide vaccine of Streptococcus pneumoniae in patients with nephrotic syndrome. Twenty-three normal subjects and 19 patients with nephrotic syndrome were vaccinated with tridecavalent pneumococcal capsular polysaccharide vaccine of Streptococcus pneumoniae. The antibody response of the nephrotic patients to pneumococcal capsular antigens was equal to that of the control subjects. These findings indicate that patients with nephrotic syndrome, despite hypogammaglobulinemia, can mount an adequate antibody response to pneumococcal polysaccharides and that there is no evidence of suppressor thymus-derived (T) cells of dysfunctioning bone marrow-derived (B) cells in these patients. | [
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PMID:26727 | Vulnerability of keto bile acids to alkaline hydrolysis. | Rigorous alkaline hydrolysis of the two primary (cholic and chenodeoxycholic) and of the two preponderant secondary (deoxycholic and lithocholic) bile acids found in bile led to excellent recoveries. Such was not the case with 11 different keto bile acid standards. Recoveries for a number of standards were unacceptably low and a variety of artefactural products were tentatively identified by gas-liquid chromatography. Keto bile acids bearing a keto gropu on C-3 were particularly vulnerable. In view of thee findings, quantitative and qualitative data reported on biological specimens submitted to saponification in ethanol, methanol, or even in water are of questionable significance. | Vulnerability of keto bile acids to alkaline hydrolysis. Rigorous alkaline hydrolysis of the two primary (cholic and chenodeoxycholic) and of the two preponderant secondary (deoxycholic and lithocholic) bile acids found in bile led to excellent recoveries. Such was not the case with 11 different keto bile acid standards. Recoveries for a number of standards were unacceptably low and a variety of artefactural products were tentatively identified by gas-liquid chromatography. Keto bile acids bearing a keto gropu on C-3 were particularly vulnerable. In view of thee findings, quantitative and qualitative data reported on biological specimens submitted to saponification in ethanol, methanol, or even in water are of questionable significance. | [
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PMID:26728 | Binding of [3H]ouabain to split frog skin: the role of the Na,K-ATPase in the generation of short circuit current. | The binding of [3H]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (Isc) was concurrently monitored. Both ouabain binding and Isc inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Isc inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Isc. Upon separation of the skins into two groups based upon initial Isc(Isci)--high, greater than 20 microamperemeter/cm2 and low, less than 10 microamperemeter/cm2, we observed two distinct populations. The high Isci skins bound very little ouabain before inhibition of Isc whereas low Isci skins bound one-half of the total number of sites before exhibiting any inhibition of Isc. These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Isc, and (b) at low Isc, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Isc relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: (a) increased intracellular K/Na concentrations, whether achieved through the addition of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and (b) ouabain binding, either with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number of ouabain molecules bound to the frog skins was not correlated with their initial Isc values, indicating that the spontaneous skin-to-skin variation in Isc was not related to the number of functional pump sites but, rather, to their turnover rate. | Binding of [3H]ouabain to split frog skin: the role of the Na,K-ATPase in the generation of short circuit current. The binding of [3H]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (Isc) was concurrently monitored. Both ouabain binding and Isc inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Isc inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Isc. Upon separation of the skins into two groups based upon initial Isc(Isci)--high, greater than 20 microamperemeter/cm2 and low, less than 10 microamperemeter/cm2, we observed two distinct populations. The high Isci skins bound very little ouabain before inhibition of Isc whereas low Isci skins bound one-half of the total number of sites before exhibiting any inhibition of Isc. These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Isc, and (b) at low Isc, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Isc relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: (a) increased intracellular K/Na concentrations, whether achieved through the addition of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and (b) ouabain binding, either with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number of ouabain molecules bound to the frog skins was not correlated with their initial Isc values, indicating that the spontaneous skin-to-skin variation in Isc was not related to the number of functional pump sites but, rather, to their turnover rate. | [
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PMID:26730 | Preferences by deer mice for solutions of sugars, salts, and acids in Richter-type drinking tests. | Deer mice (Peromyscus maniculatus) were tested for taste preferences in 48-hour, Richter-type drinking tests (sapid solution versus distilled water). The Ss, which were seven male and seven female wild adults, were individually housed within an environmental chamber. Test solutions were concentrations of five sugars and three salts (.005 M to 1.0 M) and of two acids (4.0 pH to 1.5 pH). The molar range was too limited to identify the most preferred concentration of glucose, but peak preferences for the other common sugars fell within the range. Preference was shown for hypotonic NaCl concentrations, but other salts and both acids were nonpreferred in all tests. Comparisons were made between these findings and results from Richter-type tests with other murine rodents. | Preferences by deer mice for solutions of sugars, salts, and acids in Richter-type drinking tests. Deer mice (Peromyscus maniculatus) were tested for taste preferences in 48-hour, Richter-type drinking tests (sapid solution versus distilled water). The Ss, which were seven male and seven female wild adults, were individually housed within an environmental chamber. Test solutions were concentrations of five sugars and three salts (.005 M to 1.0 M) and of two acids (4.0 pH to 1.5 pH). The molar range was too limited to identify the most preferred concentration of glucose, but peak preferences for the other common sugars fell within the range. Preference was shown for hypotonic NaCl concentrations, but other salts and both acids were nonpreferred in all tests. Comparisons were made between these findings and results from Richter-type tests with other murine rodents. | [
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PMID:26734 | Effect on the Cushing response of different rates of expansion of a supratentorial mass. | The effects on the three components (respiration, blood pressure, and heart rate) of the Cushing response (CR) were studied in cats by the continuous expansion of a supratentorial balloon. The rate of expansion was varied over the range of 0.006 to 0.6 ml/min, during whic systemic arterial pressure, heart rate, respiratory rate, and blood gases were monitored. For the different rates the time the CR took to develop, and the balloon volume required for that development were measured. The final volume ("critical volume") for eliciting the CR was more or less constant over the full range of rates of infusion (balloon expansion), a fact that supports that Monro-Kellie doctrine. This constancy of critical volume (CCV) gives rise to a highly statistically significant relationship between the rate of infusion and the latency to the production of the CR, and it is described by a power curve. Thus the development of cerebral dysfunction under these experimental conditions is independent of the rate of expansion and only dependent upon this critical volume. Exceptions to this concept of a critical volume, at the extreme of rates of expansion of lesions in patients, are predicted. | Effect on the Cushing response of different rates of expansion of a supratentorial mass. The effects on the three components (respiration, blood pressure, and heart rate) of the Cushing response (CR) were studied in cats by the continuous expansion of a supratentorial balloon. The rate of expansion was varied over the range of 0.006 to 0.6 ml/min, during whic systemic arterial pressure, heart rate, respiratory rate, and blood gases were monitored. For the different rates the time the CR took to develop, and the balloon volume required for that development were measured. The final volume ("critical volume") for eliciting the CR was more or less constant over the full range of rates of infusion (balloon expansion), a fact that supports that Monro-Kellie doctrine. This constancy of critical volume (CCV) gives rise to a highly statistically significant relationship between the rate of infusion and the latency to the production of the CR, and it is described by a power curve. Thus the development of cerebral dysfunction under these experimental conditions is independent of the rate of expansion and only dependent upon this critical volume. Exceptions to this concept of a critical volume, at the extreme of rates of expansion of lesions in patients, are predicted. | [
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PMID:26735 | Bacterial etiology of otitis media during the first six weeks of life. | Tympanocentesis was performed on 70 infants who had otitis media during the first six weeks of life. The bacteria isolated from their middle-ear effusions were Streptococcus pneumoniae (13 patients), Neisseria catarrhalis (11 patients), Hemophilus influenzae (ten patients), Enterobacteriaceae (four patients), Staphylococcus aureus (four patients), streptococci (groups A and B) (three patients), and Pseudomonas aeruginosa (two patients). Thirty patients (42.9%) had middle-ear effusions which did not contain pathogenic bacteria. Twenty-seven infants were followed for at least 12 months and 12 (44.4%) of these infants had six or more episodes of otitis media during the observation period. Further studies will be needed to establish the significance of middle-ear disease at this age and the role of therapy in improving its outcome. | Bacterial etiology of otitis media during the first six weeks of life. Tympanocentesis was performed on 70 infants who had otitis media during the first six weeks of life. The bacteria isolated from their middle-ear effusions were Streptococcus pneumoniae (13 patients), Neisseria catarrhalis (11 patients), Hemophilus influenzae (ten patients), Enterobacteriaceae (four patients), Staphylococcus aureus (four patients), streptococci (groups A and B) (three patients), and Pseudomonas aeruginosa (two patients). Thirty patients (42.9%) had middle-ear effusions which did not contain pathogenic bacteria. Twenty-seven infants were followed for at least 12 months and 12 (44.4%) of these infants had six or more episodes of otitis media during the observation period. Further studies will be needed to establish the significance of middle-ear disease at this age and the role of therapy in improving its outcome. | [
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PMID:26736 | In vivo and in vitro excystation of Zygocotyle lunata (Trematoda) metacercariae and histochemical observations on the cyst. | Encysted metacercariae of Zygocotyle lunata (Trematoda) excyst within 2 hr postexposure in the lower ileum of the domestic chick. Optimal in vitro excystation of this species occurs following pretreatment of the cyst for 15 min in 1% acidified pepsin, treatment in 0.02 M sodium dithionite (a reductant) for 1 to 2 min and then 2 hr treatment in an excystation medium containing 1% sodium glycocholate plus 1% trypsin in Earle's BSS adjusted to pH 8.8 with tris and maintained at 41 C. The cyst of this species is a dome-shaped hemisphere containing an inner and outer wall. The outer wall contains mainly acid mucopolysaccharides, whereas the inner wall is mainly proteinaceous. The cyst contains a ventral lid which only was visualized during excystation. | In vivo and in vitro excystation of Zygocotyle lunata (Trematoda) metacercariae and histochemical observations on the cyst. Encysted metacercariae of Zygocotyle lunata (Trematoda) excyst within 2 hr postexposure in the lower ileum of the domestic chick. Optimal in vitro excystation of this species occurs following pretreatment of the cyst for 15 min in 1% acidified pepsin, treatment in 0.02 M sodium dithionite (a reductant) for 1 to 2 min and then 2 hr treatment in an excystation medium containing 1% sodium glycocholate plus 1% trypsin in Earle's BSS adjusted to pH 8.8 with tris and maintained at 41 C. The cyst of this species is a dome-shaped hemisphere containing an inner and outer wall. The outer wall contains mainly acid mucopolysaccharides, whereas the inner wall is mainly proteinaceous. The cyst contains a ventral lid which only was visualized during excystation. | [
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PMID:26737 | Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine. | Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients. | Studies with Brugia pahangi 17. The anthelmintic effects of diethylcarbamazine. Diethylcarbamazine (DEC) was active in vitro against infective larvae and microfilariae of Brugia pahangi but only at high concentrations. When fed to mosquitoes which were infected with B. pahangi it had little or no activity. In jirds it was inactive against B. pahangi microfilariae and adults when administered at 300 mg/kg for 5 days either by the intraperitoneal or oral route. In cats given 25 or 50 mg DEC/kg intraperitoneally on 3 or 5 occasions it was not microfilaricidal, but most of the adult worms died within 30 days of the end of treatment. Although most microfilariae disappeared from the blood of cats immediately (i.e., within an hour) after treatment, they reappeared within a few hours in the same numbers. Microfilarial levels were reduced after treatment but there was no precipitate decline as occurs in human B. malayi patients. | [
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PMID:26739 | Tryptamine-induced drug effects insensitive to serotoninergic antagonists: evidence of specific tryptaminergic receptor stimulation? | The drug effects of tryptamine and 5-hydroxytryptopham (5-HTP) in the rabbit were compared following monoamine oxidase inhibition and various drug pretreatments. Both agents evoked hyperthermia and behavioural excitation; tryptamine but not 5-HTP also produced forepaw clonic activity. Serotoninergic receptor blockers abolished the effects of 5-HTP but only weakly influenced tryptamine responses. Both tryptamine and 5-HTP effects were potentiated by fluoxetine. Methergoline, a putative tryptaminergic receptor blocker, antagonized tryptamine-induced hyperthermia and forepaw clonus but did not influence 5-HTP responses. It is postulated that while 5-HTP produces its effects through a serotoninergic mechanism, some of the responses to tryptamine result from activation of a specific tryptamine-sensitive mechanism. | Tryptamine-induced drug effects insensitive to serotoninergic antagonists: evidence of specific tryptaminergic receptor stimulation? The drug effects of tryptamine and 5-hydroxytryptopham (5-HTP) in the rabbit were compared following monoamine oxidase inhibition and various drug pretreatments. Both agents evoked hyperthermia and behavioural excitation; tryptamine but not 5-HTP also produced forepaw clonic activity. Serotoninergic receptor blockers abolished the effects of 5-HTP but only weakly influenced tryptamine responses. Both tryptamine and 5-HTP effects were potentiated by fluoxetine. Methergoline, a putative tryptaminergic receptor blocker, antagonized tryptamine-induced hyperthermia and forepaw clonus but did not influence 5-HTP responses. It is postulated that while 5-HTP produces its effects through a serotoninergic mechanism, some of the responses to tryptamine result from activation of a specific tryptamine-sensitive mechanism. | [
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PMID:26740 | Rectal absorption of homatropine [14C]methylbromide in the rat. | Homatropine[14C]methylbromide (HMB-14C) was administered to rats by intramuscular injection, oral gavage and rectal suppository. Plasma concentration of 14C were measured over the subsequent 12 h. Peak plasma concentrations were higher and achieved more rapidly after rectal administration than by the other routes whether HMB-14C was administered in a water-soluble suppository base or in aqueous solution. Twelve h after the suppositories were inserted and retained 28% of the 14C had been excreted in the urine while 56% remained in the large intestine. Unlabelled HMB, given in rectal suppositories to anaesthetized rats, caused prompt blockade of the effects of vagal stimulation on pulse rate and of intravenous acetylcholine on blood pressure. These results confirm the rapid rectal absorption of the drug. | Rectal absorption of homatropine [14C]methylbromide in the rat. Homatropine[14C]methylbromide (HMB-14C) was administered to rats by intramuscular injection, oral gavage and rectal suppository. Plasma concentration of 14C were measured over the subsequent 12 h. Peak plasma concentrations were higher and achieved more rapidly after rectal administration than by the other routes whether HMB-14C was administered in a water-soluble suppository base or in aqueous solution. Twelve h after the suppositories were inserted and retained 28% of the 14C had been excreted in the urine while 56% remained in the large intestine. Unlabelled HMB, given in rectal suppositories to anaesthetized rats, caused prompt blockade of the effects of vagal stimulation on pulse rate and of intravenous acetylcholine on blood pressure. These results confirm the rapid rectal absorption of the drug. | [
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PMID:26741 | The mechanism of the hypothermic effect of amantadine in rats and mice. | Amantadine (25--100 mg kg-1, i.p.) given to rats at an ambient temperature of 4 degrees, or mice at 21 degrees, caused a marked fall in rectal temperature. Prior administration of pimozide (1--2 mg kg-1, s.c.) did not block hypothermia due to amantadine in rats or mice; in contrast, hypothermia due to apomorphine (2 mg kg-1, i.p.) and piribedil (10--40 mg kg-1, i.p.) in rats was blocked by pimozide pretreatment. Amphetamine (5 mg kg-1, i.p.) given 2 h after reserpine (2 mg kg-1, i.p.) caused a reversal of the hypothermic effect of reserpine in mice, but a reversal was not obtained with amantadine (50 mg kg-1, i.p.). Direct injection of amantadine (4--8 mg kg-1) into the cerebral ventricles (i.c.v.) of mice caused marked hypothermia which was not blocked by pimozide, but intravenous injection of the same dose of amantadine did not cause hypothermia. Rimantadine, a congener of amantadine but without anti-parkinsonian activity, also caused pimozide insensitive hypothermia in mice at doses of 50 mg kg-1, intraperitoneally or 2--4 mg kg-1, intracerebroventricularly. The main conclusion drawn from these results is that in causing hypothermia amantadine acts in the cns but not on dopamine receptors. | The mechanism of the hypothermic effect of amantadine in rats and mice. Amantadine (25--100 mg kg-1, i.p.) given to rats at an ambient temperature of 4 degrees, or mice at 21 degrees, caused a marked fall in rectal temperature. Prior administration of pimozide (1--2 mg kg-1, s.c.) did not block hypothermia due to amantadine in rats or mice; in contrast, hypothermia due to apomorphine (2 mg kg-1, i.p.) and piribedil (10--40 mg kg-1, i.p.) in rats was blocked by pimozide pretreatment. Amphetamine (5 mg kg-1, i.p.) given 2 h after reserpine (2 mg kg-1, i.p.) caused a reversal of the hypothermic effect of reserpine in mice, but a reversal was not obtained with amantadine (50 mg kg-1, i.p.). Direct injection of amantadine (4--8 mg kg-1) into the cerebral ventricles (i.c.v.) of mice caused marked hypothermia which was not blocked by pimozide, but intravenous injection of the same dose of amantadine did not cause hypothermia. Rimantadine, a congener of amantadine but without anti-parkinsonian activity, also caused pimozide insensitive hypothermia in mice at doses of 50 mg kg-1, intraperitoneally or 2--4 mg kg-1, intracerebroventricularly. The main conclusion drawn from these results is that in causing hypothermia amantadine acts in the cns but not on dopamine receptors. | [
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PMID:26742 | Some pharmacological actions of acetylsecohemicholinium. | The effects of the acetylated derivative of HC-3 (acetylsecohemicholinium; AcHC-3) have been studied at cholinergic nerve terminals and compared with the effects of the parent compound. AcHC-3 blocked neuromuscular transmission in nerve-muscle preparations; it was shown to be less effective than HC-3 in producing a pre-junctional block in the rat diaphragm but was more effective than HC-3 in eliciting a post-junctional blocking effect in the chick biventer muscle. On the frog rectus abdominis muscle AcHC-3 caused a substantial potentiation of the contractures elicited by acetylcholine but did not by itself cause a contracture of the muscle. AcHC-3 inhibited the synthesis of acetylcholine by cholinergic nerve ending particles and inhibited the uptake of [14C]choline into brain synaptosomal fractions to a similar extent to HC-3. AcHC-3 was shown to be a substrate for cholinesterase enzymes although the rate of hydrolysis was much less than the rate of hydrolysis of acetylcholine. It is concluded that AcHC-3 is effective in inhibiting cholinergic transmission and this action is exerted by the open chain (seco) compound and is not due to the hydrolysis of the AcHC-3 by cholinesterases to form the active HC-3 molecule. | Some pharmacological actions of acetylsecohemicholinium. The effects of the acetylated derivative of HC-3 (acetylsecohemicholinium; AcHC-3) have been studied at cholinergic nerve terminals and compared with the effects of the parent compound. AcHC-3 blocked neuromuscular transmission in nerve-muscle preparations; it was shown to be less effective than HC-3 in producing a pre-junctional block in the rat diaphragm but was more effective than HC-3 in eliciting a post-junctional blocking effect in the chick biventer muscle. On the frog rectus abdominis muscle AcHC-3 caused a substantial potentiation of the contractures elicited by acetylcholine but did not by itself cause a contracture of the muscle. AcHC-3 inhibited the synthesis of acetylcholine by cholinergic nerve ending particles and inhibited the uptake of [14C]choline into brain synaptosomal fractions to a similar extent to HC-3. AcHC-3 was shown to be a substrate for cholinesterase enzymes although the rate of hydrolysis was much less than the rate of hydrolysis of acetylcholine. It is concluded that AcHC-3 is effective in inhibiting cholinergic transmission and this action is exerted by the open chain (seco) compound and is not due to the hydrolysis of the AcHC-3 by cholinesterases to form the active HC-3 molecule. | [
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PMID:26743 | A mammalian muscle with the pharmacological characteristics of slow tonic muscle. | The guinea-pig isolated cremaster muscle gave tetrodotoxin-resistant dose-related contractures with acetylcholine, carbachol and the depolarizing neuromuscular blocking agents. The dose-response curve to suxamethonium in tetrodotoxin 2 X 10(-7)M could be shifted to the right with tubocurarine 10(-6)M. KCl, 0.1M, produced slow sustained contractures of the muscle. With the cremaster nerve-muscle preparation tetanic contractions at 20 Hz were maintained over several minutes. Tetrodotoxin eliminated the twitch response to single shock nerve stimulation but not the sustained increase in tension produced by suxamethonium. The results suggest either that there is a component of slow tonic muscle in the guinea-pig cremaster or that the cremaster consists of a type of focally innervated muscle which has pharmacological responses qualitatively different from those of most focally innervated muscles so far described. | A mammalian muscle with the pharmacological characteristics of slow tonic muscle. The guinea-pig isolated cremaster muscle gave tetrodotoxin-resistant dose-related contractures with acetylcholine, carbachol and the depolarizing neuromuscular blocking agents. The dose-response curve to suxamethonium in tetrodotoxin 2 X 10(-7)M could be shifted to the right with tubocurarine 10(-6)M. KCl, 0.1M, produced slow sustained contractures of the muscle. With the cremaster nerve-muscle preparation tetanic contractions at 20 Hz were maintained over several minutes. Tetrodotoxin eliminated the twitch response to single shock nerve stimulation but not the sustained increase in tension produced by suxamethonium. The results suggest either that there is a component of slow tonic muscle in the guinea-pig cremaster or that the cremaster consists of a type of focally innervated muscle which has pharmacological responses qualitatively different from those of most focally innervated muscles so far described. | [
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PMID:26744 | Effect of carbenoxolone on lipolysis in rat adipose tissue. | Carbenoxolone slightly but significantly decreased the release of FFA from rat epididymal fat pads. The antilipolytic action of carbenoxolone was not blocked by 10(-3)M 3-isobutyl-1-methylxanthine, a potent inhibitor of phosphodiesterase. The findings suggest that carbenoxolone exerts its antilipolytic activity by acting on adenylate cyclase, thereby decreasing cyclic AMP concentrations and the activity of the hormone-sensitive lipase in adipose tissue. | Effect of carbenoxolone on lipolysis in rat adipose tissue. Carbenoxolone slightly but significantly decreased the release of FFA from rat epididymal fat pads. The antilipolytic action of carbenoxolone was not blocked by 10(-3)M 3-isobutyl-1-methylxanthine, a potent inhibitor of phosphodiesterase. The findings suggest that carbenoxolone exerts its antilipolytic activity by acting on adenylate cyclase, thereby decreasing cyclic AMP concentrations and the activity of the hormone-sensitive lipase in adipose tissue. | [
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PMID:26745 | The inhibition by clorgyline of 5-hydroxytryptamine deamination by the rat liver. | The inhibition of the monoamine oxidase activity in the rat liver by the substrate selective inhibitor clorgyline has been investigated with 5-hydroxytryptamine as substrate. The results obtained are consistent with a theoretical model whereby the inhibition of enzyme activity by clorgyline follows a reversible association phase leading to an irreversible 'suicide' reaction. The relative concentrations of enzyme and inhibitor are of the same order, and can account both for the failure of the reaction to go to completion, and for the differences in the apparent sensitivity of enzyme preparations to inhibition by clorgyline. The possible value of this type of inhibition as a means of assay for monoamine oxidase active centres is discussed. | The inhibition by clorgyline of 5-hydroxytryptamine deamination by the rat liver. The inhibition of the monoamine oxidase activity in the rat liver by the substrate selective inhibitor clorgyline has been investigated with 5-hydroxytryptamine as substrate. The results obtained are consistent with a theoretical model whereby the inhibition of enzyme activity by clorgyline follows a reversible association phase leading to an irreversible 'suicide' reaction. The relative concentrations of enzyme and inhibitor are of the same order, and can account both for the failure of the reaction to go to completion, and for the differences in the apparent sensitivity of enzyme preparations to inhibition by clorgyline. The possible value of this type of inhibition as a means of assay for monoamine oxidase active centres is discussed. | [
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PMID:26761 | Interaction of cortisol-21-palmitate with liposomes examined by differential scanning calorimetry. | Liposomes have been suggested as carriers for corticosteroids in the local treatment of arthritis by intra-articular injection. The long chain 21-esters of cortisol such as the palmitate or octanoate are taken up and retained by liposomes in higher concentration than cortisol itself. Differential scanning calorimetry has been used to show that the cortisol ester is anchored in the liposome phospholipid bilayer by the acyl side chain. In addition, the limiting concentration of cortisol-21-palmitate which can be incorporated into dipalmitolyl phosphatidylcholine liposomes has been measured by observing changes in the DSC spectrum at different steroid concentrations. Steroid in excess of this concentration limit forms a separate phase which can be identified by nuclear magnetic resonance. For optimum effect, the treatment of arthritis with liposomes must be carried out with liposomes containing steroid below the limiting concentration. | Interaction of cortisol-21-palmitate with liposomes examined by differential scanning calorimetry. Liposomes have been suggested as carriers for corticosteroids in the local treatment of arthritis by intra-articular injection. The long chain 21-esters of cortisol such as the palmitate or octanoate are taken up and retained by liposomes in higher concentration than cortisol itself. Differential scanning calorimetry has been used to show that the cortisol ester is anchored in the liposome phospholipid bilayer by the acyl side chain. In addition, the limiting concentration of cortisol-21-palmitate which can be incorporated into dipalmitolyl phosphatidylcholine liposomes has been measured by observing changes in the DSC spectrum at different steroid concentrations. Steroid in excess of this concentration limit forms a separate phase which can be identified by nuclear magnetic resonance. For optimum effect, the treatment of arthritis with liposomes must be carried out with liposomes containing steroid below the limiting concentration. | [
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PMID:26762 | The measurement of the adhesion of film coatings to tablet surfaces: the effect of tablet porosity, surface roughness and film thickness. | The effect of tablet porosity, surface roughness and film thickness on the adhesion of hydroxypropyl methyl cellulose films to placebo tablet substrates have been studied using a specially designed tensile tester (Fisher & Rowe, 4976). There were direct relations between measured adhesion and tablet porosity and also surface roughness and tablet porosity. The effect of film thickness on the measured adhesion is complex with an initial decrease with thicknesses up to 35 micron and then a gradual increase with thicknesses up to 140 micron dut to differences in the strees distribution within the film during testing. A knowledge of these effects is necessary if results from various sources are to be compared. The findings illustrate the potential capability of the extrapolation of measured adhesion results to zero porosity and zero thickness values in order to obtain a measure of the true or intrinsic adhesion at any film/tablet interface without the confusing elements of tablet porosity, surface roughness and residual stresses in the film. | The measurement of the adhesion of film coatings to tablet surfaces: the effect of tablet porosity, surface roughness and film thickness. The effect of tablet porosity, surface roughness and film thickness on the adhesion of hydroxypropyl methyl cellulose films to placebo tablet substrates have been studied using a specially designed tensile tester (Fisher & Rowe, 4976). There were direct relations between measured adhesion and tablet porosity and also surface roughness and tablet porosity. The effect of film thickness on the measured adhesion is complex with an initial decrease with thicknesses up to 35 micron and then a gradual increase with thicknesses up to 140 micron dut to differences in the strees distribution within the film during testing. A knowledge of these effects is necessary if results from various sources are to be compared. The findings illustrate the potential capability of the extrapolation of measured adhesion results to zero porosity and zero thickness values in order to obtain a measure of the true or intrinsic adhesion at any film/tablet interface without the confusing elements of tablet porosity, surface roughness and residual stresses in the film. | [
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PMID:26763 | On methods of expressing dissolution rate data. | The value of the Weibull, logarithmic-logistic, and logarithmic-normal plots in expressing dissolution rate data is considered for the combinations of zero and first-order release with sink and non-sink conditions, and for actual dissolution rate data of diazepam from tablets in a medium of pH2. | On methods of expressing dissolution rate data. The value of the Weibull, logarithmic-logistic, and logarithmic-normal plots in expressing dissolution rate data is considered for the combinations of zero and first-order release with sink and non-sink conditions, and for actual dissolution rate data of diazepam from tablets in a medium of pH2. | [
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PMID:26765 | Correlation of physicochemical properties with absorption and metabolism of some tricyclic drugs. | Octanol and dodecane partition coefficients, surface activity and adsorbability to activated charcoal were determined for six tricyclic psychotropic drugs with N-dimethylalkyl side chains. Surface activity correlated well with the partition coefficients, and all drugs obeyed the Langmuir adsorption isotherm. A correlation between the reciprocal of the death time of gold fish exposed to drugs and partition coefficients was observed. The extent to which the drugs were N-demethylated as measured by formaldehyde formed in rat liver homogenate incubations correlated with their adsorbability to activated charcoal but not with their ability to inhibit aniline-p-hydroxylase, nor was there a linear correspondence between N-DEMETHYLATION AND DRUG LIPOPHILICITY AS INDICATed by partition coefficients or surface activity. | Correlation of physicochemical properties with absorption and metabolism of some tricyclic drugs. Octanol and dodecane partition coefficients, surface activity and adsorbability to activated charcoal were determined for six tricyclic psychotropic drugs with N-dimethylalkyl side chains. Surface activity correlated well with the partition coefficients, and all drugs obeyed the Langmuir adsorption isotherm. A correlation between the reciprocal of the death time of gold fish exposed to drugs and partition coefficients was observed. The extent to which the drugs were N-demethylated as measured by formaldehyde formed in rat liver homogenate incubations correlated with their adsorbability to activated charcoal but not with their ability to inhibit aniline-p-hydroxylase, nor was there a linear correspondence between N-DEMETHYLATION AND DRUG LIPOPHILICITY AS INDICATed by partition coefficients or surface activity. | [
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PMID:26766 | Investigation of possible interactions between substance P and transmitter mechanisms in the substantia nigra and corpus striatum of the rat. | The effect of substnace P (SP) on the uptake and release of radiolabelled dopamine (3H-DA), 5-hydroxytryptamine (3H-5-HT) and y-aminobutyric acid (3H-GABA) was studied in slices of rat substantia nigra and corpus triatum. SP, 10(-9) to 10(-5) m, failed to modify the uptakes of these compounds during incubations (10-90 min) with slices of either brain region. SP, 10(-6)M, had no apparent effect on the spontaneous output of any of these compounds in either substantia nigra or corpus striatum. In the corpus striatum, SP seemed to potentiate the potassium-stimulated outflow of 3H-DA and 3H-5-HT, but not 3H-GABA, while the realeases from substantia nigra were unaffected. Morphine (10(-3)M), but not met-enkephalin (5 X 10(-6)M), weakly antagonized K+- EVOKED RELEASE OF 3/-DA in the corpus striatum. These results are discussed with reference to the possible interaction of SP with transmitter mechanisms at presynaptic sites in the central nervous system. | Investigation of possible interactions between substance P and transmitter mechanisms in the substantia nigra and corpus striatum of the rat. The effect of substnace P (SP) on the uptake and release of radiolabelled dopamine (3H-DA), 5-hydroxytryptamine (3H-5-HT) and y-aminobutyric acid (3H-GABA) was studied in slices of rat substantia nigra and corpus triatum. SP, 10(-9) to 10(-5) m, failed to modify the uptakes of these compounds during incubations (10-90 min) with slices of either brain region. SP, 10(-6)M, had no apparent effect on the spontaneous output of any of these compounds in either substantia nigra or corpus striatum. In the corpus striatum, SP seemed to potentiate the potassium-stimulated outflow of 3H-DA and 3H-5-HT, but not 3H-GABA, while the realeases from substantia nigra were unaffected. Morphine (10(-3)M), but not met-enkephalin (5 X 10(-6)M), weakly antagonized K+- EVOKED RELEASE OF 3/-DA in the corpus striatum. These results are discussed with reference to the possible interaction of SP with transmitter mechanisms at presynaptic sites in the central nervous system. | [
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PMID:26767 | Quisqualamine, a novel gamma-aminobutyric acid (GABA) related depressant amino acid. | A new substnace, quisqualamine, the decarboxylated analogue of quisqualic acid, predictably depressed electrical activity of neurons of the frog and rat spinal cord in vitro and of the mouse spinal cord in vivo. In the in vitro preparations, the action of quisqualamine was associated with a prolonged depolarization of primary afferent terminals which was sensitive to blockade by picrotoxin and bicuculline and which was also depressed by strychnine. This suggests an interaction of quisqualamine with presynaptic receptors for both GABA and beta-alanine. Post-synaptic actions of quisqualamine, which were less marked than those at presynaptic sites, also appeared to be predominantly GABA-mimetic in vitro, though a sensitivity to the GABA-antagonist bicuculline could not be demonstrated in vitro. | Quisqualamine, a novel gamma-aminobutyric acid (GABA) related depressant amino acid. A new substnace, quisqualamine, the decarboxylated analogue of quisqualic acid, predictably depressed electrical activity of neurons of the frog and rat spinal cord in vitro and of the mouse spinal cord in vivo. In the in vitro preparations, the action of quisqualamine was associated with a prolonged depolarization of primary afferent terminals which was sensitive to blockade by picrotoxin and bicuculline and which was also depressed by strychnine. This suggests an interaction of quisqualamine with presynaptic receptors for both GABA and beta-alanine. Post-synaptic actions of quisqualamine, which were less marked than those at presynaptic sites, also appeared to be predominantly GABA-mimetic in vitro, though a sensitivity to the GABA-antagonist bicuculline could not be demonstrated in vitro. | [
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PMID:26768 | Rat brain and serum lithium concentrations after acute injections of lithium carbonate and orotate. | Eight hours after intraperitoneal injections of 1.0, 2.0, and 4.0m equiv Li kg-1, the serum and brain lithium concentrations of rats were significantly greater after lithium orotate than after lithium carbonate. While little serum lithium remained at 24 h after injection of 2.0 m equiv kg-1 lithium carbonate, two-thirds of the 2 h serum lithium concentration was present 24h after lithium orotate. Furthermore, the 24 h brain concentration of lithium after lithium orotate was approximately three times greater than that after lithium carbonate. These data suggest the possibility that lower doses of lithium orotate than lithium carbonate may achieve therapeutic brain lithium concentrations and relatively stable serum concentrations. | Rat brain and serum lithium concentrations after acute injections of lithium carbonate and orotate. Eight hours after intraperitoneal injections of 1.0, 2.0, and 4.0m equiv Li kg-1, the serum and brain lithium concentrations of rats were significantly greater after lithium orotate than after lithium carbonate. While little serum lithium remained at 24 h after injection of 2.0 m equiv kg-1 lithium carbonate, two-thirds of the 2 h serum lithium concentration was present 24h after lithium orotate. Furthermore, the 24 h brain concentration of lithium after lithium orotate was approximately three times greater than that after lithium carbonate. These data suggest the possibility that lower doses of lithium orotate than lithium carbonate may achieve therapeutic brain lithium concentrations and relatively stable serum concentrations. | [
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PMID:26769 | Corticosterone concentrations in mice during ethanol drinking and withdrawal. | Consumption of an ethanol-containing diet by mice resulted in a significant increase in circulating concentrations of corticosterone which was maintained for 8 days. There were no changes in the concentrations of plasma corticosterone binding globulin. Ethanol withdrawal symptoms followed the removal of ethanol from the diet and circulating corticosterone concentrations were further increased. There was no correlation between blood ethanol and glucocorticoid concentrations during the chronic ethanol treatment. Stress related to ethanol consumption may be of greater importance than the circulating ethanol concentrations in producing the elevation in plasma glucocorticoids. | Corticosterone concentrations in mice during ethanol drinking and withdrawal. Consumption of an ethanol-containing diet by mice resulted in a significant increase in circulating concentrations of corticosterone which was maintained for 8 days. There were no changes in the concentrations of plasma corticosterone binding globulin. Ethanol withdrawal symptoms followed the removal of ethanol from the diet and circulating corticosterone concentrations were further increased. There was no correlation between blood ethanol and glucocorticoid concentrations during the chronic ethanol treatment. Stress related to ethanol consumption may be of greater importance than the circulating ethanol concentrations in producing the elevation in plasma glucocorticoids. | [
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PMID:26770 | Antidotal effects of dimethyl sulphoxide against paracetamol-, bromobenzene-, and thioacetamide-induced hepatotoxicity. | In mice the hepatotoxic effects of paracetamol (0.5-1.0 g kg-1, orally) as evidenced by increased serum enzyme activities of the aminotransferases and sorbitol dehydrogenase were dose-dependently inhibited by simultaneous treatment with dimethyl sulphoxide (DMSO 0,25-1.0 g kg-1, i.p.). DMSO was also active against bromobenzene- and thioacetamide-induced hepatotoxicity, but failed to protect mice against carbon tetrachloride-induced liver damage. Hepatic glutathione depletion in mice amounting to 94% after paracetamol (0.5 g kg-1, orally) and to 60% after bromobezene (0.25 ml kg-1, orally) was dose-dependently reduced by the simultaneous administration of DMSO(0.25--1.0 G KG-1, I.P.). This indicates less conjugation of the toxic metabolites of paracetamol and bromobenzene to liver glutathione (G-SH) in the presence of DMSO. | Antidotal effects of dimethyl sulphoxide against paracetamol-, bromobenzene-, and thioacetamide-induced hepatotoxicity. In mice the hepatotoxic effects of paracetamol (0.5-1.0 g kg-1, orally) as evidenced by increased serum enzyme activities of the aminotransferases and sorbitol dehydrogenase were dose-dependently inhibited by simultaneous treatment with dimethyl sulphoxide (DMSO 0,25-1.0 g kg-1, i.p.). DMSO was also active against bromobenzene- and thioacetamide-induced hepatotoxicity, but failed to protect mice against carbon tetrachloride-induced liver damage. Hepatic glutathione depletion in mice amounting to 94% after paracetamol (0.5 g kg-1, orally) and to 60% after bromobezene (0.25 ml kg-1, orally) was dose-dependently reduced by the simultaneous administration of DMSO(0.25--1.0 G KG-1, I.P.). This indicates less conjugation of the toxic metabolites of paracetamol and bromobenzene to liver glutathione (G-SH) in the presence of DMSO. | [
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PMID:26786 | Rearrangement of N-benzyl-2-cyano-2-(hydroxyimino)acetamide. | The reduction of N-benzyl-2-cyano-2-(hydroxyimino)acetamide resulted in the formation of N-benzyl-1,2-ethanediamine and N-benzyl-N'-methyl-1,2-ethanediamine in approximately an equimolar ratio. The formation of the two unexpected products is explained by the migration of a cyano group in a Beckmann-type rearrangement. | Rearrangement of N-benzyl-2-cyano-2-(hydroxyimino)acetamide. The reduction of N-benzyl-2-cyano-2-(hydroxyimino)acetamide resulted in the formation of N-benzyl-1,2-ethanediamine and N-benzyl-N'-methyl-1,2-ethanediamine in approximately an equimolar ratio. The formation of the two unexpected products is explained by the migration of a cyano group in a Beckmann-type rearrangement. | [
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PMID:26787 | Interaction of tricyclic antipsychotic and antidepressant drugs with 1-anilino-8-naphthalenesulfonic acid. | The binding of 1-anilino-8-naphthalenesulfonic acid to selected tricyclic antipsychotic and antidepressant drugs was studied by fluorescence spectroscopy. The acid exhibited an increase in fluorescence intensity accompanied by a hypsochromic shift of the emission lambdamax in the presence of these drugs. These fluorescence characteristics, in addition to those of acid-drug complexes after addition of potassium chloride or urea, suggested that binding was hydrophobic. The spectra also provided evidence regarding the importance of certain structural features of drugs in determining the nature of binding. | Interaction of tricyclic antipsychotic and antidepressant drugs with 1-anilino-8-naphthalenesulfonic acid. The binding of 1-anilino-8-naphthalenesulfonic acid to selected tricyclic antipsychotic and antidepressant drugs was studied by fluorescence spectroscopy. The acid exhibited an increase in fluorescence intensity accompanied by a hypsochromic shift of the emission lambdamax in the presence of these drugs. These fluorescence characteristics, in addition to those of acid-drug complexes after addition of potassium chloride or urea, suggested that binding was hydrophobic. The spectra also provided evidence regarding the importance of certain structural features of drugs in determining the nature of binding. | [
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PMID:26788 | Solubility studies of silver sulfonamides. | The solubilities of silver sulfapyridine, silver sulfamethazine, and silver sulfamethizole as a function of pH were determined in nitric acid-potassium nitrate, acetate, and sulfonic acid buffers. All silver sulfonamides showed an increase in solubility with increasing hydrogen-ion concentration, a behavior which closely paralleled the protonation of the p-amino function of the sulfonamide. A silver-ion selective electrode was used to measure silver-ion concentration in solution and the methods of known subtraction and known addition were used to measure total silver. Both silver sulfamethizole and silver sulfamethazine were ionized completely in solution. Silver sulfapyridine was ionized completely only in the more acidic pH 2-3 range. A comparison of the physical properties of the silver salts for which mortality studies were available revealed a unique set of properties for silver sulfadiazine. | Solubility studies of silver sulfonamides. The solubilities of silver sulfapyridine, silver sulfamethazine, and silver sulfamethizole as a function of pH were determined in nitric acid-potassium nitrate, acetate, and sulfonic acid buffers. All silver sulfonamides showed an increase in solubility with increasing hydrogen-ion concentration, a behavior which closely paralleled the protonation of the p-amino function of the sulfonamide. A silver-ion selective electrode was used to measure silver-ion concentration in solution and the methods of known subtraction and known addition were used to measure total silver. Both silver sulfamethizole and silver sulfamethazine were ionized completely in solution. Silver sulfapyridine was ionized completely only in the more acidic pH 2-3 range. A comparison of the physical properties of the silver salts for which mortality studies were available revealed a unique set of properties for silver sulfadiazine. | [
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PMID:26789 | Ketonic oxidation products of cyclobarbital. | Chemical or biochemical oxidation of cyclobarbital yielded 5-(3-oxo-1-cyclohexen-1-yl)-5-ethyl -2,4,6,- (1H,3H,5H)-pyrimidinetrione, and photochemical oxidation gave 5-(6-oxo-1-cyclohexen-1-yl)-5-ethyl-2,4,6-(1H,3H,3H)-pyrimidinetrione. These compounds were isolated and purified by TLC, and their structures were determined by UV, IR, NMR, and mass spectral data; the crystalline structure was determined by X-ray diffraction. | Ketonic oxidation products of cyclobarbital. Chemical or biochemical oxidation of cyclobarbital yielded 5-(3-oxo-1-cyclohexen-1-yl)-5-ethyl -2,4,6,- (1H,3H,5H)-pyrimidinetrione, and photochemical oxidation gave 5-(6-oxo-1-cyclohexen-1-yl)-5-ethyl-2,4,6-(1H,3H,3H)-pyrimidinetrione. These compounds were isolated and purified by TLC, and their structures were determined by UV, IR, NMR, and mass spectral data; the crystalline structure was determined by X-ray diffraction. | [
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PMID:26790 | Colorimetric determination of nadolol in tablets. | Nadolol was extracted from tablet excipients with an acidic potassium chloride solution. The drug was oxidized with periodic acid, and the resulting aldehyde was reacted with 2,4-dinitrophenylhydrazine to form the corresponding hydrazone. Excess reagent was removed with a cupric chloride solution. The hydrazone was extracted into chloroform, and its absorbance was measured at the 352-nm maximum. | Colorimetric determination of nadolol in tablets. Nadolol was extracted from tablet excipients with an acidic potassium chloride solution. The drug was oxidized with periodic acid, and the resulting aldehyde was reacted with 2,4-dinitrophenylhydrazine to form the corresponding hydrazone. Excess reagent was removed with a cupric chloride solution. The hydrazone was extracted into chloroform, and its absorbance was measured at the 352-nm maximum. | [
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PMID:26791 | Possible ion-pair-mediated absorption of mixidine I: partitioning and lethality studies. | Mixidine, a very soluble base which is completely ionized in all physiological fluids, was found to form ion-pairs as demonstrated by its ability to partition into 1-butanol from acidic solutions. A similar relationship was observed for the effect of acids on the absorption of intraduodenally and orally administered solutions of mixidine in rats (as determined by lethality). Studies also demonstrated that the pH-lethality effects were not specific for a particular counterion. Mixidine was more lethal when administered intraduodenally than when administered orally, and the counterions per se were not lethal in the doses used. | Possible ion-pair-mediated absorption of mixidine I: partitioning and lethality studies. Mixidine, a very soluble base which is completely ionized in all physiological fluids, was found to form ion-pairs as demonstrated by its ability to partition into 1-butanol from acidic solutions. A similar relationship was observed for the effect of acids on the absorption of intraduodenally and orally administered solutions of mixidine in rats (as determined by lethality). Studies also demonstrated that the pH-lethality effects were not specific for a particular counterion. Mixidine was more lethal when administered intraduodenally than when administered orally, and the counterions per se were not lethal in the doses used. | [
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PMID:26792 | Possible ion-pair-mediated absorption of mixidine II: plasma levels and histology. | Plasma intact 14C-mixidine levels in rats increased when the drug was administered intraduodenally with 1:3 and 1:5 molar ratios of 2-naphthalenesulfonic acid. Upon histological examination of the duodenums, similar doses of mixidine combined with 2-naphthalenesulfonic acid produced no dose-related lesions. These and previous observations demonstrate that mixidine absorption may be enhanced by ion-pair formation. | Possible ion-pair-mediated absorption of mixidine II: plasma levels and histology. Plasma intact 14C-mixidine levels in rats increased when the drug was administered intraduodenally with 1:3 and 1:5 molar ratios of 2-naphthalenesulfonic acid. Upon histological examination of the duodenums, similar doses of mixidine combined with 2-naphthalenesulfonic acid produced no dose-related lesions. These and previous observations demonstrate that mixidine absorption may be enhanced by ion-pair formation. | [
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PMID:26793 | Simple GLC analysis of anticonvulsant drugs in commercial dosage forms. | A simple, specific GLC procedure is described for the analysis of one sedative and six anticonvulsant drugs in pharmaceutical dosage forms. Sample aliquots of ethotoin, glutethimide, mephenytoin, methsuximide, and phensuximide were shaken with or extracted into ethyl acetate, diluted with the internal standard (diphenyl phthalate) solution, injected into a gas chromatograph, and eluted from a methylsilicon column. Primidone and phenytoin samples (extracted as the free acid) required derivatization with N,O-bis(trimethylsilyl)acetamide prior to chromatography. The same temperature programming conditions and flow rate settings were used for all seven drugs. The GLC results agreed well with those obtained using the pharmacopeial methods. | Simple GLC analysis of anticonvulsant drugs in commercial dosage forms. A simple, specific GLC procedure is described for the analysis of one sedative and six anticonvulsant drugs in pharmaceutical dosage forms. Sample aliquots of ethotoin, glutethimide, mephenytoin, methsuximide, and phensuximide were shaken with or extracted into ethyl acetate, diluted with the internal standard (diphenyl phthalate) solution, injected into a gas chromatograph, and eluted from a methylsilicon column. Primidone and phenytoin samples (extracted as the free acid) required derivatization with N,O-bis(trimethylsilyl)acetamide prior to chromatography. The same temperature programming conditions and flow rate settings were used for all seven drugs. The GLC results agreed well with those obtained using the pharmacopeial methods. | [
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PMID:26794 | Effects of patulin on the kinetics of substrate and cationic ligand activation of adenosine triphosphatase in mouse brain. | Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one), a carcinogenic lactone produced as a major metabolite by several fungi, inhibited the Mg++-dependent Na+-K+ activated adenosine triphosphatase (ATPase) activity of mouse brain microsomal fractions with an estimated IC50 of 3.0 X 10(-4) M. Inhibition was concentration dependent. Hydrolysis of ATP was linear with both time and enzyme concentration either with or without patulin in reaction mixtures. Altered pH and activity curves for Na+-K+ ATPase demonstrated comparable inhibition by patulin in buffered acidic ranges through an optimum of 7.5, followed by a reduction of toxicity to this system at higher alkaline pH. Kinetic studies of cationic-substrate activation of Na+-K+ ATPase indicated noncompetitive inhibition with respect to ATP (at low affinity nucleotide-directed sites) and Na+ (in the presence of low, noninterfering concentrations of K+). Competitive inhibition with respect to activation of the Na+-k+-stimulated activity and K+-stimulated p-nitrophenyl phosphatase activity of the enzyme system was indicated by altered binding site parameters without change in apparent Vmax in the presence of patulin. Activity was partially restored by washing. Preincubation of patulin with dithiothreitol or glutathione protected the enzyme from inhibition. Results suggest that patulin exerted its effect on Na+-K+ ATPase either directly by interfering with K+ binding or indirectly by inducing a conformational change in the enzyme. | Effects of patulin on the kinetics of substrate and cationic ligand activation of adenosine triphosphatase in mouse brain. Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one), a carcinogenic lactone produced as a major metabolite by several fungi, inhibited the Mg++-dependent Na+-K+ activated adenosine triphosphatase (ATPase) activity of mouse brain microsomal fractions with an estimated IC50 of 3.0 X 10(-4) M. Inhibition was concentration dependent. Hydrolysis of ATP was linear with both time and enzyme concentration either with or without patulin in reaction mixtures. Altered pH and activity curves for Na+-K+ ATPase demonstrated comparable inhibition by patulin in buffered acidic ranges through an optimum of 7.5, followed by a reduction of toxicity to this system at higher alkaline pH. Kinetic studies of cationic-substrate activation of Na+-K+ ATPase indicated noncompetitive inhibition with respect to ATP (at low affinity nucleotide-directed sites) and Na+ (in the presence of low, noninterfering concentrations of K+). Competitive inhibition with respect to activation of the Na+-k+-stimulated activity and K+-stimulated p-nitrophenyl phosphatase activity of the enzyme system was indicated by altered binding site parameters without change in apparent Vmax in the presence of patulin. Activity was partially restored by washing. Preincubation of patulin with dithiothreitol or glutathione protected the enzyme from inhibition. Results suggest that patulin exerted its effect on Na+-K+ ATPase either directly by interfering with K+ binding or indirectly by inducing a conformational change in the enzyme. | [
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PMID:26795 | Beta adrenergic blockade, regional left ventricular blood flow and ST-segment elevation in canine experimental myocardial ischemia. | The effects of dl-propranolol, d-propranolol, dl-pindolol and dl-practolol on regional myocardial blood flow (assessed by means of tracer microspheres) and on ST-segment elevation in ischemic and nonischemic areas of the canine left ventricle have been investigated. dl-Propranolol and dl-pindolol, but not dl-practolol and d-propranolol, induced blood flow redistribution from the epicardium to the endocardium both in ischemic and nonischemic areas. dl-Propranolol-induced redistribution was abolished by atrial pacing at the control heart rate value. These results indicate that the redistribution phenomenon only occurs if both a bradycardia-inducing beta1 adrenoreceptor blockade and a coronary vessels beta2 adrenoceptor blockade are simultaneously achieved. All four drugs significantly decreased ST-segment elevation in ischemic areas. Under atrial pacing, this effect was abolished with dl-practolol but only reduced with dl- and d-propranol, suggesting that, besides bradycardia, membrane stabilization might be involved in protection against ST-segment elevation in ischemic areas. | Beta adrenergic blockade, regional left ventricular blood flow and ST-segment elevation in canine experimental myocardial ischemia. The effects of dl-propranolol, d-propranolol, dl-pindolol and dl-practolol on regional myocardial blood flow (assessed by means of tracer microspheres) and on ST-segment elevation in ischemic and nonischemic areas of the canine left ventricle have been investigated. dl-Propranolol and dl-pindolol, but not dl-practolol and d-propranolol, induced blood flow redistribution from the epicardium to the endocardium both in ischemic and nonischemic areas. dl-Propranolol-induced redistribution was abolished by atrial pacing at the control heart rate value. These results indicate that the redistribution phenomenon only occurs if both a bradycardia-inducing beta1 adrenoreceptor blockade and a coronary vessels beta2 adrenoceptor blockade are simultaneously achieved. All four drugs significantly decreased ST-segment elevation in ischemic areas. Under atrial pacing, this effect was abolished with dl-practolol but only reduced with dl- and d-propranol, suggesting that, besides bradycardia, membrane stabilization might be involved in protection against ST-segment elevation in ischemic areas. | [
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PMID:26797 | Liberation of cyanide from alpha-aminonitriles relative to amygdalin. | A series of aminoitriles have been synthesized and studied whose nonenzymatic dissociation with release of cyanide may be varied by modest alteration of their molecular structure from that obtained with nonenzymatic dissociation of amygdalin to that obtained from enzymatic dissociation of amygdalin by substantial quantities of beta-glucosidase. The relationship between such alterations in molecular structure and nonenzymatic dissociation is discussed. A combination of the results of these studies and studies relating molecular structure to physical localization propensity in tumors has potential in the design of chemotherapeutic agents. | Liberation of cyanide from alpha-aminonitriles relative to amygdalin. A series of aminoitriles have been synthesized and studied whose nonenzymatic dissociation with release of cyanide may be varied by modest alteration of their molecular structure from that obtained with nonenzymatic dissociation of amygdalin to that obtained from enzymatic dissociation of amygdalin by substantial quantities of beta-glucosidase. The relationship between such alterations in molecular structure and nonenzymatic dissociation is discussed. A combination of the results of these studies and studies relating molecular structure to physical localization propensity in tumors has potential in the design of chemotherapeutic agents. | [
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PMID:26798 | Inhibition of sympathetic nervous system by histamine:studies with H1- and H2-receptor antagonists. | A study was designed to investigate the effect of histamine on sympathetic transmission to the myocardium in pentobarbital-anesthetized dogs. Intravenous infusion of histamine (1.0 and 2.0 microgram/kg/min) produced dose-related decreases in blood pressure and caused significant impairment of cardioacceleration observed during stimulation of the right postganglionic cardiac sympathetic nerve fibers. Positive chronotropic effects of intravenously administered norepinephrine as well as tyramine were not altered during histamine infusion. Blockade of neuronal reuptake with desipramine did not modify the inhibitory action of histamine on sympathetic nerve function. Prior administration of mepyramine (pyrilamine), a histamine type H1-receptor antagonist caused partial attenuation of the depressor action of histamine, but did not prevent histamine-induced inhibition of neurogenic function. Further treatment with metiamide, a histamine type H2-receptor antagonist caused nearly complete attenuation of the depressor response to histamine and also significantly antagonized the inhibitory action of histamine on sympathetic transmission to the myocardium. It is concluded that while the depressor action of histamine is due to the activation of both H1- as well as H2-receptors, histamine causes impairment of sympathetic nerve function to the myocardium by acting on H2-receptors which may be located on sympathetic nerve terminals. | Inhibition of sympathetic nervous system by histamine:studies with H1- and H2-receptor antagonists. A study was designed to investigate the effect of histamine on sympathetic transmission to the myocardium in pentobarbital-anesthetized dogs. Intravenous infusion of histamine (1.0 and 2.0 microgram/kg/min) produced dose-related decreases in blood pressure and caused significant impairment of cardioacceleration observed during stimulation of the right postganglionic cardiac sympathetic nerve fibers. Positive chronotropic effects of intravenously administered norepinephrine as well as tyramine were not altered during histamine infusion. Blockade of neuronal reuptake with desipramine did not modify the inhibitory action of histamine on sympathetic nerve function. Prior administration of mepyramine (pyrilamine), a histamine type H1-receptor antagonist caused partial attenuation of the depressor action of histamine, but did not prevent histamine-induced inhibition of neurogenic function. Further treatment with metiamide, a histamine type H2-receptor antagonist caused nearly complete attenuation of the depressor response to histamine and also significantly antagonized the inhibitory action of histamine on sympathetic transmission to the myocardium. It is concluded that while the depressor action of histamine is due to the activation of both H1- as well as H2-receptors, histamine causes impairment of sympathetic nerve function to the myocardium by acting on H2-receptors which may be located on sympathetic nerve terminals. | [
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PMID:26800 | The role of peripheral catecholamines in oxotremorine tremor in the rat and its antagonism by beta adrenoceptor blocking agents. | Oxotremorine, 0.25 mg/kg, produces marked tremor in the rat, which is abolished by scopolamine, 0.5 mg/kg, and is substantially reduced in intensity and duration both by adrenalmedullectomy and by chemical sympathectomy with 6-hydroxydopamine. Oxotremorine increases plasma norepinephrine from 0.62 +/- 0.07 to 3.01 +/- 0.47 ng/ml and plasma epinephrine, from 0.82 +/- 0.14 to 3.42 +/- 0.48 ng/ml, in conscious unrestrained rats. l-Propranolol (0.5-2.5 mg/kg) reduces tremor, and at 2.5 mg/kg is more effective than either chemical sympathectomy or adrenal demedullation. d-Propranolol and sotalol are also active at 4 and 10 times the dose of l-propranolol, respectively. l-Propranolol does not prevent the rise in catecholamines induced by oxotremorine. It is suggested that stimulation of central muscarinic receptors causes tremor by a combination of two effects. There is an increase in cholinergic influence to motor efferents accompanied by an activation of the sympathoadrenal system to release catecholamines which augment tremor by stimulation of beta2 adrenoceptors. | The role of peripheral catecholamines in oxotremorine tremor in the rat and its antagonism by beta adrenoceptor blocking agents. Oxotremorine, 0.25 mg/kg, produces marked tremor in the rat, which is abolished by scopolamine, 0.5 mg/kg, and is substantially reduced in intensity and duration both by adrenalmedullectomy and by chemical sympathectomy with 6-hydroxydopamine. Oxotremorine increases plasma norepinephrine from 0.62 +/- 0.07 to 3.01 +/- 0.47 ng/ml and plasma epinephrine, from 0.82 +/- 0.14 to 3.42 +/- 0.48 ng/ml, in conscious unrestrained rats. l-Propranolol (0.5-2.5 mg/kg) reduces tremor, and at 2.5 mg/kg is more effective than either chemical sympathectomy or adrenal demedullation. d-Propranolol and sotalol are also active at 4 and 10 times the dose of l-propranolol, respectively. l-Propranolol does not prevent the rise in catecholamines induced by oxotremorine. It is suggested that stimulation of central muscarinic receptors causes tremor by a combination of two effects. There is an increase in cholinergic influence to motor efferents accompanied by an activation of the sympathoadrenal system to release catecholamines which augment tremor by stimulation of beta2 adrenoceptors. | [
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PMID:26804 | Synthesis and pharmacological activity and some derivatives of 1-phenyl-1,2,3,4-tetrahydro-5H-1,4-benzodiazepin-5-one. | 4-N-Alkylamino derivatives and corresponding ammonium quaternary salts of tetrahydro-1,4-benzodiazepin-5-one were synthesized and evaluated for psychotropic activity in mice by ip via. This study was also extended to some nitro and amino derivatives of tetrahydro-1,4-benzodiazepin-5-one. Compounds were devoid of tranquilizing activity and in comparison with two classical benzodiazepines, chlordiazepoxide and diazepam, they showed high toxicity and little or no effect on motor coordination, motor activity, and maximal electroshock. On some "in vitro" tests the compounds exhibited pharmacological properties when they were used at high concentrations. | Synthesis and pharmacological activity and some derivatives of 1-phenyl-1,2,3,4-tetrahydro-5H-1,4-benzodiazepin-5-one. 4-N-Alkylamino derivatives and corresponding ammonium quaternary salts of tetrahydro-1,4-benzodiazepin-5-one were synthesized and evaluated for psychotropic activity in mice by ip via. This study was also extended to some nitro and amino derivatives of tetrahydro-1,4-benzodiazepin-5-one. Compounds were devoid of tranquilizing activity and in comparison with two classical benzodiazepines, chlordiazepoxide and diazepam, they showed high toxicity and little or no effect on motor coordination, motor activity, and maximal electroshock. On some "in vitro" tests the compounds exhibited pharmacological properties when they were used at high concentrations. | [
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PMID:26805 | A method for the isolation of Bacteroides melaninogenicus from the human mouth. | Isolation procedures involving the direct plating of specimens and the use of selective, non-selective, transport and enrichment media were compared in respect of their value for the recovery of Bacteroides melaninogenicus from the gingival sulcus. A selective medium containing kanamycin and vancomycin enhanced the recovery rate of B. melaninogenicus. VMGII transport medium was convenient to use and gave as good a recovery rate as that obtained with plates directly inoculated in the clinic. | A method for the isolation of Bacteroides melaninogenicus from the human mouth. Isolation procedures involving the direct plating of specimens and the use of selective, non-selective, transport and enrichment media were compared in respect of their value for the recovery of Bacteroides melaninogenicus from the gingival sulcus. A selective medium containing kanamycin and vancomycin enhanced the recovery rate of B. melaninogenicus. VMGII transport medium was convenient to use and gave as good a recovery rate as that obtained with plates directly inoculated in the clinic. | [
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PMID:26807 | Amino acid activation with adenosine 5'-phosphorimidazolide. | Amino acids are activated by reaction with adenosine 5'-phosphorimidazolide in aqueous imidazole buffers. If adenosine 5'-(O-methylphosphate), an analogue of the 3'-terminus of t-RNA is present, 2'(3')-O-aminoacyladenosine 5'-(O-methylphosphate) is formed. Fifteen percent of this compound accumulated at pH 5.8, but less was formed at higher pHs. The highest efficiency of utilization if ImpA attained in our experiments was about 24%. Analogous reactions occured with several other amino acids, including a number that have functional side-chains. | Amino acid activation with adenosine 5'-phosphorimidazolide. Amino acids are activated by reaction with adenosine 5'-phosphorimidazolide in aqueous imidazole buffers. If adenosine 5'-(O-methylphosphate), an analogue of the 3'-terminus of t-RNA is present, 2'(3')-O-aminoacyladenosine 5'-(O-methylphosphate) is formed. Fifteen percent of this compound accumulated at pH 5.8, but less was formed at higher pHs. The highest efficiency of utilization if ImpA attained in our experiments was about 24%. Analogous reactions occured with several other amino acids, including a number that have functional side-chains. | [
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PMID:26808 | Intracellular electrolytes in erythrocytes during and after shock: relation to impaired consciousness. | Of 32 patients in shock and catabolic state following shock with impaired consciousness 31 exhibited raised sodium content in their erythrocytes. On the assumption that the erythrocyte membrane acts the same as nerve cell membrane, the hypofunction of erythrocyte membrane may result in impaired consciousness. The hypofunction of erythrocyte membrane is assumed by its increased permeability in shock. A positive osmotic discrepancy between measured and calculated levels denotes altered membranous permeability. Subjectively, impaired consciousness was evaluated by clinical grading. Meanwhile, as a trial of quantification of conscious levels, we applied a new technique of analysis of power spectrum obtained by computer on the autocorrelogram of the EEG during intermittent photic stimulation. This new analytic method was useful in evaluating objective changes of cerebral function. There was a good correlation between raised sodium content in erythrocytes and depressed power spectrum. The degree of increased sodium in erythrocytes seems to correlate with patients' clinical prognosis. | Intracellular electrolytes in erythrocytes during and after shock: relation to impaired consciousness. Of 32 patients in shock and catabolic state following shock with impaired consciousness 31 exhibited raised sodium content in their erythrocytes. On the assumption that the erythrocyte membrane acts the same as nerve cell membrane, the hypofunction of erythrocyte membrane may result in impaired consciousness. The hypofunction of erythrocyte membrane is assumed by its increased permeability in shock. A positive osmotic discrepancy between measured and calculated levels denotes altered membranous permeability. Subjectively, impaired consciousness was evaluated by clinical grading. Meanwhile, as a trial of quantification of conscious levels, we applied a new technique of analysis of power spectrum obtained by computer on the autocorrelogram of the EEG during intermittent photic stimulation. This new analytic method was useful in evaluating objective changes of cerebral function. There was a good correlation between raised sodium content in erythrocytes and depressed power spectrum. The degree of increased sodium in erythrocytes seems to correlate with patients' clinical prognosis. | [
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PMID:26809 | Transfection of Streptococcus pneumoniae with bacteriophage DNA. | It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966). | Transfection of Streptococcus pneumoniae with bacteriophage DNA. It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966). | [
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PMID:26810 | Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. | We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins. | Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins. | [
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PMID:26811 | Lymphangiography in patients with malignancy in a non-descended testicle. | Lymphangiography was performed on 23 patients with malignancy in non-descended testicles, 14 of whom had seminomatous and 9 non-seminomatous tumors. Lymph node metastases were shown by lymphangiography in 8 patients: 3 in lumbar nodes, 1 in iliac nodes alone and 4 in lumbar and iliac lymph nodes. Microscopic metastases were shown in lumbar nodes at retroperitoneal lymph node dissection in 2 patients when the lymphangiograms were negative. Iliac lymph node metastases are rare in testicular tumors but may be seen in tumors of non-descended testicles, alone or in combination with lumbar metastatic disease. This information is extremely important for the radiologist as well as the clinician in the management of patients. | Lymphangiography in patients with malignancy in a non-descended testicle. Lymphangiography was performed on 23 patients with malignancy in non-descended testicles, 14 of whom had seminomatous and 9 non-seminomatous tumors. Lymph node metastases were shown by lymphangiography in 8 patients: 3 in lumbar nodes, 1 in iliac nodes alone and 4 in lumbar and iliac lymph nodes. Microscopic metastases were shown in lumbar nodes at retroperitoneal lymph node dissection in 2 patients when the lymphangiograms were negative. Iliac lymph node metastases are rare in testicular tumors but may be seen in tumors of non-descended testicles, alone or in combination with lumbar metastatic disease. This information is extremely important for the radiologist as well as the clinician in the management of patients. | [
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PMID:26812 | Screening urography in asymptomatic cryptorchid patients. | Excretory urograms in 140 cryptorchid patients free of associated urologic symptoms are reviewed. Our findings are presented and compared to those of previously reported series. Our results do not indicate a specific indication for the routine use of excretory urography in asymptomatic male subjects with cryptorchidism. | Screening urography in asymptomatic cryptorchid patients. Excretory urograms in 140 cryptorchid patients free of associated urologic symptoms are reviewed. Our findings are presented and compared to those of previously reported series. Our results do not indicate a specific indication for the routine use of excretory urography in asymptomatic male subjects with cryptorchidism. | [
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PMID:26813 | Anorchism versus cryptorchidism: the importance of a diligent search for intra-abdominal testes. | We reviewed 13 cases of testicular tumors arising in cryptorchid, intra-abdominal testes. All types of germinal neoplasms were found, seminoma being the most common. Of the 13 patients 5 had undergone limited exploration previously, with no testis being identified and, thus, were thought to have had unilateral anorchism. The importance of a thorough exploration of the peritoneal cavity is emphasized. | Anorchism versus cryptorchidism: the importance of a diligent search for intra-abdominal testes. We reviewed 13 cases of testicular tumors arising in cryptorchid, intra-abdominal testes. All types of germinal neoplasms were found, seminoma being the most common. Of the 13 patients 5 had undergone limited exploration previously, with no testis being identified and, thus, were thought to have had unilateral anorchism. The importance of a thorough exploration of the peritoneal cavity is emphasized. | [
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PMID:26814 | Prostatic utricle cysts (müllerian duct cysts). | A series of 88 male patients with prostatic utricle cysts (müllerian duct) has been compiled by adding our 3 patients to 85 cases reported. Irritative lower urinary tract symptoms were the most common presenting complaint (42 per cent), while lower obstructive symptoms were noted in 29 per cent. Of the 20 clinical case reports in boys 25 per cent presented with epididymitis often in an undescended gonad. A cystic rectal mass found in half of the patients represents the most common presenting sign, while hypospadias occurred in a fourth. Unilateral renal dysgenesis or agenesis was associated in 10 per cent of the reports. While the suprapubic surgical approach to excision has been used most commonly it has proved unsatisfactory in two-thirds of the cases. The posterior approach was used successfully in 2 boys with preservation of erectile potency in both. The wide spectrum of histopathology of these cysts prevents clear characterization. A 3% incidence of malignancy in prostatic utricle cysts is noted. | Prostatic utricle cysts (müllerian duct cysts). A series of 88 male patients with prostatic utricle cysts (müllerian duct) has been compiled by adding our 3 patients to 85 cases reported. Irritative lower urinary tract symptoms were the most common presenting complaint (42 per cent), while lower obstructive symptoms were noted in 29 per cent. Of the 20 clinical case reports in boys 25 per cent presented with epididymitis often in an undescended gonad. A cystic rectal mass found in half of the patients represents the most common presenting sign, while hypospadias occurred in a fourth. Unilateral renal dysgenesis or agenesis was associated in 10 per cent of the reports. While the suprapubic surgical approach to excision has been used most commonly it has proved unsatisfactory in two-thirds of the cases. The posterior approach was used successfully in 2 boys with preservation of erectile potency in both. The wide spectrum of histopathology of these cysts prevents clear characterization. A 3% incidence of malignancy in prostatic utricle cysts is noted. | [
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PMID:26815 | Reconstruction of the urinary tract in prune belly uropathy. | Ten patients with prune bell uropathy, including 7 neonates, were treated with extensive surgical tailoring of the upper urinary tracts using primarily the upper ureteral segment. Simultaneous transabdominal orchiopexy, particularly in the neonate, is described as a useful adjunct. Our early results in these patients justify this aggressive approach. | Reconstruction of the urinary tract in prune belly uropathy. Ten patients with prune bell uropathy, including 7 neonates, were treated with extensive surgical tailoring of the upper urinary tracts using primarily the upper ureteral segment. Simultaneous transabdominal orchiopexy, particularly in the neonate, is described as a useful adjunct. Our early results in these patients justify this aggressive approach. | [
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] |
PMID:26818 | Treatment of the agitated patient with an organic brain disorder. | Agitated patients with organic brain disorders represent relatively common diagnostic and management problems. Therapeutic failures usually result from a failure to understand the patient's disturbed behavior, the staff's emotional response to the patient's behavior, or neglect of the biological cause and ineffective use of medication. Effective management depends on continued monitoring of the patient's mental status and physical condition. | Treatment of the agitated patient with an organic brain disorder. Agitated patients with organic brain disorders represent relatively common diagnostic and management problems. Therapeutic failures usually result from a failure to understand the patient's disturbed behavior, the staff's emotional response to the patient's behavior, or neglect of the biological cause and ineffective use of medication. Effective management depends on continued monitoring of the patient's mental status and physical condition. | [
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PMID:26821 | [Comparative examinations of gastric juice in healthy infants and in infants suffering from enteritis (author's transl)]. | There are significant differences between the shapes of curves of gastric juice volume, acid quantity, pH levels, and peptic activity, in the three age groups of healthy infants under examination. Significant differences are also seen in all three age groups with regard to the quantity of acid calculated in relation to 1 ml gastric juice, as well as the relative peptic activity (volume activity). The amount of gastric juice is proportional to the bodyweight and body surface of the infants, whereas acid concentration and peptic volume activity show a relatively steeper increase. | [Comparative examinations of gastric juice in healthy infants and in infants suffering from enteritis (author's transl)]. There are significant differences between the shapes of curves of gastric juice volume, acid quantity, pH levels, and peptic activity, in the three age groups of healthy infants under examination. Significant differences are also seen in all three age groups with regard to the quantity of acid calculated in relation to 1 ml gastric juice, as well as the relative peptic activity (volume activity). The amount of gastric juice is proportional to the bodyweight and body surface of the infants, whereas acid concentration and peptic volume activity show a relatively steeper increase. | [
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