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PMID:25813 | A new modification of colpocleisis for treatment of total procidentia in old age. | A new approach for treating uterine prolapse in old age is presented. Fiftten patients underwent this operation during our 5-year study. The procedure is simple and brief and can be performed, using a local anesthetic, on patients whose general condition is poor. The patients in our series had no immediate postoperative complications, and those who attended our follow-up clinic had no recurrence of the disease. | A new modification of colpocleisis for treatment of total procidentia in old age. A new approach for treating uterine prolapse in old age is presented. Fiftten patients underwent this operation during our 5-year study. The procedure is simple and brief and can be performed, using a local anesthetic, on patients whose general condition is poor. The patients in our series had no immediate postoperative complications, and those who attended our follow-up clinic had no recurrence of the disease. | [
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PMID:25814 | Study of "spontaneous" abortion in Thailand. | This analysis includes data on 3 530 patients with septic or incomplete, inevitable or threatened abortions who were treated at the Siriraj Hospital in Bangkok between January 1972 and December 1973. Data on the patients' socio-demographic characteristics; their reproductive, abortion, and medical histories; their pre- and postabortion contraceptive acceptance; and a clinical profile of their abortion treatment, including procedure time, length of hospitalization, and immediate and delayed postoperative complications are analyzed. | Study of "spontaneous" abortion in Thailand. This analysis includes data on 3 530 patients with septic or incomplete, inevitable or threatened abortions who were treated at the Siriraj Hospital in Bangkok between January 1972 and December 1973. Data on the patients' socio-demographic characteristics; their reproductive, abortion, and medical histories; their pre- and postabortion contraceptive acceptance; and a clinical profile of their abortion treatment, including procedure time, length of hospitalization, and immediate and delayed postoperative complications are analyzed. | [
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PMID:25815 | The cold sterilization of abortion cannulae. | The purpose of this study was to determine if cold sterilization is a feasible method for sterilizing abortion cannulae and, if so, to find the appropriate solutions and time intervals for this sterilization process. Study findings show that abortion cannulae can be cold sterilized by soaking them for a minimum of 10 minutes in a solution of Cidex or 95% ethanol. Soaking the cannulae for 20 minutes in a 2% tincture of iodine solution also appears to be useful for decontamination purposes. | The cold sterilization of abortion cannulae. The purpose of this study was to determine if cold sterilization is a feasible method for sterilizing abortion cannulae and, if so, to find the appropriate solutions and time intervals for this sterilization process. Study findings show that abortion cannulae can be cold sterilized by soaking them for a minimum of 10 minutes in a solution of Cidex or 95% ethanol. Soaking the cannulae for 20 minutes in a 2% tincture of iodine solution also appears to be useful for decontamination purposes. | [
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PMID:25816 | Extramembranous pregnancy: a case report. | A rare case of extramembranous pregnancy is reported. It was associated with intermittent loss of liquor and bloody discharge beginning at about the 25th week of gestation. An anencephalic infant was delivered alive from a breech presentation during the 31st week of gestation. The placenta was markedly circumvallate, with short, thick membranes which did not cover more than 10% of the infant. The course of the pregnancy and the examination of the infant and placenta are discussed, and the pertinent literature is reviewed. | Extramembranous pregnancy: a case report. A rare case of extramembranous pregnancy is reported. It was associated with intermittent loss of liquor and bloody discharge beginning at about the 25th week of gestation. An anencephalic infant was delivered alive from a breech presentation during the 31st week of gestation. The placenta was markedly circumvallate, with short, thick membranes which did not cover more than 10% of the infant. The course of the pregnancy and the examination of the infant and placenta are discussed, and the pertinent literature is reviewed. | [
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PMID:25820 | Management of acute anxiety syndrome with parenterally administered lorazepam. | After the preliminary successful use of injectable lorazepam in calming 20 patients who presented with acute anxiety crises, a formal study of 115 other such patients was carried out. All were seen either in a hospital emergency room or on an emergency out-patient basis in private practice. Treatment consisted of an initial intravenous injection of lorazepam (3mg) followed, if necessary, by up to three further injections within a 24-hour period. The result was usually dramatic: complete abolition or reasonable control of symptoms within 30 minutes in all but 2 patients. The major effect was relaxant and sedative; 62 patients slept following the injections. although 55 could be easily aroused, 7 could not. Of the other patients, 49 remained awake but relaxed; only 4 remained tense and were regarded as treatment failures. No significant side-effects or changes in vital signs were noted. The results support and extend those reported by other invesigators in a recent controlled, double-blind (but otherwise similarly conducted) trial. | Management of acute anxiety syndrome with parenterally administered lorazepam. After the preliminary successful use of injectable lorazepam in calming 20 patients who presented with acute anxiety crises, a formal study of 115 other such patients was carried out. All were seen either in a hospital emergency room or on an emergency out-patient basis in private practice. Treatment consisted of an initial intravenous injection of lorazepam (3mg) followed, if necessary, by up to three further injections within a 24-hour period. The result was usually dramatic: complete abolition or reasonable control of symptoms within 30 minutes in all but 2 patients. The major effect was relaxant and sedative; 62 patients slept following the injections. although 55 could be easily aroused, 7 could not. Of the other patients, 49 remained awake but relaxed; only 4 remained tense and were regarded as treatment failures. No significant side-effects or changes in vital signs were noted. The results support and extend those reported by other invesigators in a recent controlled, double-blind (but otherwise similarly conducted) trial. | [
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PMID:25824 | [Pregnancy and labor in twins. II. Determination of twin pregnancy and avoidance of premature birth (author's transl)]. | In about 65% of the 258 twin births which occurred at the University Gynaecological Hospital, Zürich, from 1966 to 1976, it was possible to diagnose the twin pregnancy before the birth of the first twin. Twin pregnancy could be diagnosed in 96.2% of all the cases before the birth of the first twin, after routine ultrasound diagnosis of pregnancy had been introduced. Prerequisite of optimal treatment of pregnancy is a very early diagnosis which is only possible by widest use of ultrasound investigation technique. The rate of premature births was reduced from 48.0% to 27.3% by means of complete rest in bed and prophylactic tocolysis with a betamimetic from the 32nd to the 37th week of pregnancy. At the same time, the number of underweight children below 2.500 g was reduced by means of this treatment from 60.1% to 34.1%. It seems to be an undisputable fact that prolonged hospitalization in the last three months of pregnancy will improve the prognosis. | [Pregnancy and labor in twins. II. Determination of twin pregnancy and avoidance of premature birth (author's transl)]. In about 65% of the 258 twin births which occurred at the University Gynaecological Hospital, Zürich, from 1966 to 1976, it was possible to diagnose the twin pregnancy before the birth of the first twin. Twin pregnancy could be diagnosed in 96.2% of all the cases before the birth of the first twin, after routine ultrasound diagnosis of pregnancy had been introduced. Prerequisite of optimal treatment of pregnancy is a very early diagnosis which is only possible by widest use of ultrasound investigation technique. The rate of premature births was reduced from 48.0% to 27.3% by means of complete rest in bed and prophylactic tocolysis with a betamimetic from the 32nd to the 37th week of pregnancy. At the same time, the number of underweight children below 2.500 g was reduced by means of this treatment from 60.1% to 34.1%. It seems to be an undisputable fact that prolonged hospitalization in the last three months of pregnancy will improve the prognosis. | [
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PMID:25825 | [Study of H-2 mutations in mice. VII. The defectiveness of the H-2ba mutant in the hemagglutination and graft vs host reactions]. | Three mutants of the H-2K gene within the mouse major histocompatibility complex were studied. Antisera anti-H-2.5 + 39 were unable to agglutinate H-2ba and H-2bf mutant red blood cells. The same sera moderately agglutinated H-2b red blood cells. The H-2ba red blood cells did not absorb in vitro the activity from an anti-H-2.5 serum, while the H-2b cells did. Thus, these results indicate the existence of qualitative SD antigenic differences among H-2Kb derived mutants. In the proliferative graft versus host test the H-2ba spleen cells did not induce spleen enlargement in the H-2bd recipients, while pronounced reaction was recovered in bd-ba combination. The (baXb) F1 hybrid cells were as efficient as b/b homozygous cells. The data suggest that the H-2K gene product may be involved in the antigen recognition process by T cells. | [Study of H-2 mutations in mice. VII. The defectiveness of the H-2ba mutant in the hemagglutination and graft vs host reactions]. Three mutants of the H-2K gene within the mouse major histocompatibility complex were studied. Antisera anti-H-2.5 + 39 were unable to agglutinate H-2ba and H-2bf mutant red blood cells. The same sera moderately agglutinated H-2b red blood cells. The H-2ba red blood cells did not absorb in vitro the activity from an anti-H-2.5 serum, while the H-2b cells did. Thus, these results indicate the existence of qualitative SD antigenic differences among H-2Kb derived mutants. In the proliferative graft versus host test the H-2ba spleen cells did not induce spleen enlargement in the H-2bd recipients, while pronounced reaction was recovered in bd-ba combination. The (baXb) F1 hybrid cells were as efficient as b/b homozygous cells. The data suggest that the H-2K gene product may be involved in the antigen recognition process by T cells. | [
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PMID:25826 | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption. | Intestinal absorption of vitamin D-3 in physiological concentrations was studied in the live unanesthetised rat. In both the jejunum and the ileum a linear relationship was found between the absorption rate of the vitamin and its intraluminal concentration. Increasing the sodium taurocholate concentation in the perfusate above 5mM did not change ileal absorption rate but did decrease jejunal absorption rate. The vitamin's rate of absorption was raised by increases in either the hydrogen ion concentration in vivo is mediated by passive diffusion. The rate of absorption of ttion or the perfusate's flow rate. Addition of 2.5 mM fatty acids of varying chain length and degrees of saturation resulted in a decrease in the rate of vitamin D-3 absorption. These experiments indicate that vitamin D-3 absorption in vivo is mediated by passive diffusion. The rate of absorption of the vitamin is influenced by the composition of the perfusate and the thickness of the unstirred layer. | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption. Intestinal absorption of vitamin D-3 in physiological concentrations was studied in the live unanesthetised rat. In both the jejunum and the ileum a linear relationship was found between the absorption rate of the vitamin and its intraluminal concentration. Increasing the sodium taurocholate concentation in the perfusate above 5mM did not change ileal absorption rate but did decrease jejunal absorption rate. The vitamin's rate of absorption was raised by increases in either the hydrogen ion concentration in vivo is mediated by passive diffusion. The rate of absorption of ttion or the perfusate's flow rate. Addition of 2.5 mM fatty acids of varying chain length and degrees of saturation resulted in a decrease in the rate of vitamin D-3 absorption. These experiments indicate that vitamin D-3 absorption in vivo is mediated by passive diffusion. The rate of absorption of the vitamin is influenced by the composition of the perfusate and the thickness of the unstirred layer. | [
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PMID:25827 | Effects of a protein meal, intraduodenal HCl, and oleic acid on portal and peripheral venous secretin and on pancreatic bicarbonate secretion. | We have studied the effect of a protein meal on secretin (IRS) concentration in dogs and humans using a radioimmunoassay of improved sensitivity (8 pg/ml). After a meal, pancreatic bicarbonate secretion (PBS) increased markedly and proximal duodenal pH decreased from 6.2 to 4.3. Portal and peripheral IRS concentrations, however, remained unchanged in eight dogs and five patients with cirrhosis of the liver. Similarly, an alkaline solution of sodium oleate (pH 9.2) stimulated PBS but not IRS. Intraduodenal administration of various amounts of HCl in dogs demonstrated that acid-stimulated PBS was invariably accompanied by rises in peripheral venous IRS concentration. We conclude that the postprandial stimulation of PBS involves mechanisms more complex than acid-stimulated secretin release. | Effects of a protein meal, intraduodenal HCl, and oleic acid on portal and peripheral venous secretin and on pancreatic bicarbonate secretion. We have studied the effect of a protein meal on secretin (IRS) concentration in dogs and humans using a radioimmunoassay of improved sensitivity (8 pg/ml). After a meal, pancreatic bicarbonate secretion (PBS) increased markedly and proximal duodenal pH decreased from 6.2 to 4.3. Portal and peripheral IRS concentrations, however, remained unchanged in eight dogs and five patients with cirrhosis of the liver. Similarly, an alkaline solution of sodium oleate (pH 9.2) stimulated PBS but not IRS. Intraduodenal administration of various amounts of HCl in dogs demonstrated that acid-stimulated PBS was invariably accompanied by rises in peripheral venous IRS concentration. We conclude that the postprandial stimulation of PBS involves mechanisms more complex than acid-stimulated secretin release. | [
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PMID:25828 | Pathophysiological responses to meals in the Zollinger-Ellison syndrome: I. Paradoxical postprandial inhibition of gastric secretion. | The gastric acid, pepsin, and secretory volume output in response to a mixed meal were measured in six patients with Zollinger-Ellison syndrome caused by a gastrin-producing tumour proved subsequently at surgery. The patients were all normocalcaemic, and none had previous abdominal surgery. In four of the six patients, ingestion of the meal markedly inhibited the gastric secretory output, which decreased to below fasting levels, returning later to basal values. In two other patients, whose fasting acid output was considerably lower, the secretory output increased after the meal, but some inhibiton of gastric secretion was also apparent for variable intervals of time. The serum gastrin concentration in all patients remained essentially unchanged or increased after the meal. Two patients were restudied after successful removal of the duodenal gastrin-producing tumour, and in each the normal gastric secretory and gastrin-releasing responses were completely restored. Our studies suggest that, in patients with the Zollinger-Ellison syndrome caused by a gastrinoma, physiological regulatory mechanisms triggered by food reduce the continuous stimulation of gastric secretion caused by their tumoural hypergastrinaemia. | Pathophysiological responses to meals in the Zollinger-Ellison syndrome: I. Paradoxical postprandial inhibition of gastric secretion. The gastric acid, pepsin, and secretory volume output in response to a mixed meal were measured in six patients with Zollinger-Ellison syndrome caused by a gastrin-producing tumour proved subsequently at surgery. The patients were all normocalcaemic, and none had previous abdominal surgery. In four of the six patients, ingestion of the meal markedly inhibited the gastric secretory output, which decreased to below fasting levels, returning later to basal values. In two other patients, whose fasting acid output was considerably lower, the secretory output increased after the meal, but some inhibiton of gastric secretion was also apparent for variable intervals of time. The serum gastrin concentration in all patients remained essentially unchanged or increased after the meal. Two patients were restudied after successful removal of the duodenal gastrin-producing tumour, and in each the normal gastric secretory and gastrin-releasing responses were completely restored. Our studies suggest that, in patients with the Zollinger-Ellison syndrome caused by a gastrinoma, physiological regulatory mechanisms triggered by food reduce the continuous stimulation of gastric secretion caused by their tumoural hypergastrinaemia. | [
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PMID:25829 | Intestinal phase of gastric secretion in patients with duodenal ulcer. | In 10 healthy subjects and 10 duodenal ulcer patients the intestinal phase of gastric acid secretion was studied by intraduodenal infusion of a 10% liver extract meal (pH 7) at 400 ml/h for three hours. A gastroduodenal double lumen tube with two balloons was used to block the pylorus and to prevent duodenogastric reflux. Gastric acid response to a duodenal meal of liver extract reached a peak at the end of the first hour of infusion of the extract and was then followed by a relatively well-sustained plateau. When the figure was normalised as a percentage of peak response to pentagastrin it was about 45% in healthy subjects and 63% in duodenal ulcer patients. Serum gastrin concentration increased significantly during a duodenal meal of liver extract only in duodenal ulcer patients and not in healthy subjects. The combination of the duodenal meal of liver extract with pentagastrin infusion resulted in a significantly greater increase in acid output in duodenal ulcer patients than in healthy controls. Duodenal perfusion with a liver extract meal in which the pH was gradually decreased caused a pH-dependent reduction in acid output, but not in serum gastrin, both in the duodenal ulcer patients and in healthy subjects. This study shows that the intestinal phase in man results in a potent gastric acid stimulation which is pH-dependent, greatly augmented by pentagastrin, and more vigorous in duodenal ulcer patients than in healthy controls. | Intestinal phase of gastric secretion in patients with duodenal ulcer. In 10 healthy subjects and 10 duodenal ulcer patients the intestinal phase of gastric acid secretion was studied by intraduodenal infusion of a 10% liver extract meal (pH 7) at 400 ml/h for three hours. A gastroduodenal double lumen tube with two balloons was used to block the pylorus and to prevent duodenogastric reflux. Gastric acid response to a duodenal meal of liver extract reached a peak at the end of the first hour of infusion of the extract and was then followed by a relatively well-sustained plateau. When the figure was normalised as a percentage of peak response to pentagastrin it was about 45% in healthy subjects and 63% in duodenal ulcer patients. Serum gastrin concentration increased significantly during a duodenal meal of liver extract only in duodenal ulcer patients and not in healthy subjects. The combination of the duodenal meal of liver extract with pentagastrin infusion resulted in a significantly greater increase in acid output in duodenal ulcer patients than in healthy controls. Duodenal perfusion with a liver extract meal in which the pH was gradually decreased caused a pH-dependent reduction in acid output, but not in serum gastrin, both in the duodenal ulcer patients and in healthy subjects. This study shows that the intestinal phase in man results in a potent gastric acid stimulation which is pH-dependent, greatly augmented by pentagastrin, and more vigorous in duodenal ulcer patients than in healthy controls. | [
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PMID:25830 | Induction of gastro-oesophageal reflux by alcohol. | In order to establish whether alcohol in amounts in amounts customarily imbibed during social drinking causes gastro-oesophageal reflux, 12 healthy young individuals, without symptoms of gastro-oesophageal reflux, were studied twice. Each time, distal oesophageal pH was monitored continuously for three hours after a standard meal which included either 180 ml 100 proof vodka or 180 ml water. The order of studies with and without alcohol was random. Peak blood alcohol concentrations ranged between 0.63 and 1.29 g/l. Eleven of the 12 subjects refluxed more after alcohol; and the difference in mean reflux scores for studies with and without alcohol was highly significant. We conclude that relatively modest quanttities of alcohol induce gastro-oesophageal reflux in healthy people. | Induction of gastro-oesophageal reflux by alcohol. In order to establish whether alcohol in amounts in amounts customarily imbibed during social drinking causes gastro-oesophageal reflux, 12 healthy young individuals, without symptoms of gastro-oesophageal reflux, were studied twice. Each time, distal oesophageal pH was monitored continuously for three hours after a standard meal which included either 180 ml 100 proof vodka or 180 ml water. The order of studies with and without alcohol was random. Peak blood alcohol concentrations ranged between 0.63 and 1.29 g/l. Eleven of the 12 subjects refluxed more after alcohol; and the difference in mean reflux scores for studies with and without alcohol was highly significant. We conclude that relatively modest quanttities of alcohol induce gastro-oesophageal reflux in healthy people. | [
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PMID:25831 | 24-hour rhythms in uterine and umbilical blood flows of conscious pregnant sheep. | The possibility that 24-hour rhythms exist in uterine blood flow (UtBF) and umbilical blood (UmBF) was investigated in 5 days postoperative chronically instrumented near-term pregnant sheep acclimated to a controlled environment. UtBF, UmBF and pressure measurements were made at 15-min intervals over 24 h beginning at 0800 h. Each data series was examined for the presence of significant rhythms of a 24-hour period using a method of Fourier analysis. UtBF 24-hour rhythms were found in all ewes; UmBF 24-hour rhythms were found in 4 of 5 lambs. A consistent reciprocal phase relationship between UtBF and UmBF was evident within animals. There were no associated rhythms in maternal arterial, fetal arterial, or amniotic fluid pressures. These results indicate that the presence of circadian rhythms must be considered as a possible variable when long-term uteroplacental hemodynamic studies are planned. | 24-hour rhythms in uterine and umbilical blood flows of conscious pregnant sheep. The possibility that 24-hour rhythms exist in uterine blood flow (UtBF) and umbilical blood (UmBF) was investigated in 5 days postoperative chronically instrumented near-term pregnant sheep acclimated to a controlled environment. UtBF, UmBF and pressure measurements were made at 15-min intervals over 24 h beginning at 0800 h. Each data series was examined for the presence of significant rhythms of a 24-hour period using a method of Fourier analysis. UtBF 24-hour rhythms were found in all ewes; UmBF 24-hour rhythms were found in 4 of 5 lambs. A consistent reciprocal phase relationship between UtBF and UmBF was evident within animals. There were no associated rhythms in maternal arterial, fetal arterial, or amniotic fluid pressures. These results indicate that the presence of circadian rhythms must be considered as a possible variable when long-term uteroplacental hemodynamic studies are planned. | [
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PMID:25832 | [The hypertensive effect of adyston and etilefrin. Comparative animal experimental studies]. | According to the results obtained under the described conditions it can be stated that after i.v. administration Adyston induces a dose-dependent rise in systolic and diastolic blood pressure, in comparison with etilefrin the new product Adyston shows a greater potency in rising the systolic and diastolic blood pressure; Adyston induced rise of blood pressure is in general of longer duration in comparison with that induced by etilefrin, it is interesting that in the dose of 0.01 mg/kg Adyston increases the arterial blood pressure but at the same time slightly decreases the heart rate; this is not the case after administration of etilefrin which at the same dose increases both arterial blood pressure and heart rate. | [The hypertensive effect of adyston and etilefrin. Comparative animal experimental studies]. According to the results obtained under the described conditions it can be stated that after i.v. administration Adyston induces a dose-dependent rise in systolic and diastolic blood pressure, in comparison with etilefrin the new product Adyston shows a greater potency in rising the systolic and diastolic blood pressure; Adyston induced rise of blood pressure is in general of longer duration in comparison with that induced by etilefrin, it is interesting that in the dose of 0.01 mg/kg Adyston increases the arterial blood pressure but at the same time slightly decreases the heart rate; this is not the case after administration of etilefrin which at the same dose increases both arterial blood pressure and heart rate. | [
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PMID:25833 | [Treatment of parkinson's syndrome. New developments]. | The treatment of Parkinson's syndrome with newly developed drugs has to be planned within the entire therapeutic concept. Besides the anticholinergics, levodopa, amantadines and beta-blockers should be taken into consideration. When necessary, hypnotics, ataractics and antidepressant drugs should be applied. | [Treatment of parkinson's syndrome. New developments]. The treatment of Parkinson's syndrome with newly developed drugs has to be planned within the entire therapeutic concept. Besides the anticholinergics, levodopa, amantadines and beta-blockers should be taken into consideration. When necessary, hypnotics, ataractics and antidepressant drugs should be applied. | [
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PMID:25834 | Insulin effect on proteolytic activities in rat skeletal muscle. | Proteolytic activity has been measure in rat skeletal muscle by use of [14C]-hemoglobin as substrate. The activity of the alkaline proteinases increases during starvation and in diabetic state. In streptozotocin-diabetic animals the activity of alkaline proteases increases to 300% over a time of 21 days. Insulin treatment reverses the enhanced enzyme activity to normal level. | Insulin effect on proteolytic activities in rat skeletal muscle. Proteolytic activity has been measure in rat skeletal muscle by use of [14C]-hemoglobin as substrate. The activity of the alkaline proteinases increases during starvation and in diabetic state. In streptozotocin-diabetic animals the activity of alkaline proteases increases to 300% over a time of 21 days. Insulin treatment reverses the enhanced enzyme activity to normal level. | [
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PMID:25835 | The effect of stress or glucose feeding on hepatic tyrosine aminotransferase activity and liver and plasma tyrosine level of intact and adrenalectomized rats. | The increase of hepatic tyrosine aminotransferase and the fall of plasma tyrosine in rats subjected to immobilization is reconfirmed. Moreover, the same effects three hrs after exposuing the animals to 400 revolutions in Noble-Collip drums are described. However, in bilaterally adrenalectomized rats both hepatic tyrosine aminotransferase and plasma tyrosine remain unchanged after injury and the liver tyrosine level increase. Finally, in animals fed overnight exclusively with 15% glucose solution the well-known decrease of hepatic tyrosine aminotransferase was found paralleled by increased plasma tyrosine levels. A regulatory role of tyrosine aminotransferase in establishing the level of tyrosine in plasma is suggested. | The effect of stress or glucose feeding on hepatic tyrosine aminotransferase activity and liver and plasma tyrosine level of intact and adrenalectomized rats. The increase of hepatic tyrosine aminotransferase and the fall of plasma tyrosine in rats subjected to immobilization is reconfirmed. Moreover, the same effects three hrs after exposuing the animals to 400 revolutions in Noble-Collip drums are described. However, in bilaterally adrenalectomized rats both hepatic tyrosine aminotransferase and plasma tyrosine remain unchanged after injury and the liver tyrosine level increase. Finally, in animals fed overnight exclusively with 15% glucose solution the well-known decrease of hepatic tyrosine aminotransferase was found paralleled by increased plasma tyrosine levels. A regulatory role of tyrosine aminotransferase in establishing the level of tyrosine in plasma is suggested. | [
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PMID:25836 | Liver cell heterogeneity. The distribution of fructose-bisphosphatase in fed and fasted rats and in man. | Periportal and perivenous hepatocytes were isolated by microdissection from lyophilized liver slices (16 micrometer) from fed and fasted rats and from a human patient. NADP/NADPH cycling was used to determine fructose-1,6-bisphosphatase activity in the isolated hepatocytes (10 ng dry weight). The periportal hepatocytes contain 3 times as much fructose-1,6-bisphosphatase activity as the perivenous hepatocytes. A 24 h fast led to two-fold increase in the activity in the periportal hepatocytes and a four-fold increase in the perivenous hepatocytes. Fructose-1,6-bisphosphatase parallels closely with the key enzyme phosphoenolpyruvate carboxykinase, and therefore can be considered a suitable marker for gluconeogenic capacity. | Liver cell heterogeneity. The distribution of fructose-bisphosphatase in fed and fasted rats and in man. Periportal and perivenous hepatocytes were isolated by microdissection from lyophilized liver slices (16 micrometer) from fed and fasted rats and from a human patient. NADP/NADPH cycling was used to determine fructose-1,6-bisphosphatase activity in the isolated hepatocytes (10 ng dry weight). The periportal hepatocytes contain 3 times as much fructose-1,6-bisphosphatase activity as the perivenous hepatocytes. A 24 h fast led to two-fold increase in the activity in the periportal hepatocytes and a four-fold increase in the perivenous hepatocytes. Fructose-1,6-bisphosphatase parallels closely with the key enzyme phosphoenolpyruvate carboxykinase, and therefore can be considered a suitable marker for gluconeogenic capacity. | [
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PMID:25837 | Purification and characterization of aromatic-amino-acid-glyoxylate aminotransferase from monkey and rat liver. | Aromatic-amino-acid-glyoxylate aminotransferase was highly purified from the mitochondrial fraction of livers from monkey and glucagon-injected rats. The two enzyme preparations showed physical and enzymic properties different from a kynurenine aminotransferase previously described. The two enzymes had nearly identical molecular weights (approximate 80 000), isoelectric points (pH 8.0) and pH optima (pH 8.0 - 8.5). However, a difference in substrate specificity was observed between the two enzymes. Both enzymes utilized glyoxylate, pyruvate, hydroxypyruvate and 2-oxo-4-methyl-thiobutyrate as effective amino acceptors. 2-Oxoglutarate was active for rat enzyme but not for monkey enzyme. With glyoxylate, amino donors were effective in the following order of activity; phenylalanine greater than histidine greater than tyrosine greater than tryptophan greater than 5-hydroxytrypotphan greater than kynurenine for the rat enzyme, and phenylalanine greater than kynurenine greater than histidine greater than tryptophan greater than 5-hydroxy-tryptophan for the monkey enzyme. | Purification and characterization of aromatic-amino-acid-glyoxylate aminotransferase from monkey and rat liver. Aromatic-amino-acid-glyoxylate aminotransferase was highly purified from the mitochondrial fraction of livers from monkey and glucagon-injected rats. The two enzyme preparations showed physical and enzymic properties different from a kynurenine aminotransferase previously described. The two enzymes had nearly identical molecular weights (approximate 80 000), isoelectric points (pH 8.0) and pH optima (pH 8.0 - 8.5). However, a difference in substrate specificity was observed between the two enzymes. Both enzymes utilized glyoxylate, pyruvate, hydroxypyruvate and 2-oxo-4-methyl-thiobutyrate as effective amino acceptors. 2-Oxoglutarate was active for rat enzyme but not for monkey enzyme. With glyoxylate, amino donors were effective in the following order of activity; phenylalanine greater than histidine greater than tyrosine greater than tryptophan greater than 5-hydroxytrypotphan greater than kynurenine for the rat enzyme, and phenylalanine greater than kynurenine greater than histidine greater than tryptophan greater than 5-hydroxy-tryptophan for the monkey enzyme. | [
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PMID:25838 | Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes. | Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus. | Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes. Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus. | [
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PMID:25845 | Bacteriological findings in the transtracheal aspirate from patients with acute exacerbation of chronic bronchitis. | Transtracheal aspirates from 87 patients with acute exacerbations of chronic bronchitis who had received no recent antibiotic treatment were examined. A single bacterial species was found in 83% of positive cultures. Predominant pathogens were Haemophilus influenzae and Streptococcus pneumoniae which occurred jointly or separately in 50% of positive cultures. Bacteria traditionally considered as non-pathogenic for the lower respiratory tract also appeared to play an aetiological role. Enterobacteriaceae and anaerobes were infrequent. No bacterial growth was found in 11 cases. | Bacteriological findings in the transtracheal aspirate from patients with acute exacerbation of chronic bronchitis. Transtracheal aspirates from 87 patients with acute exacerbations of chronic bronchitis who had received no recent antibiotic treatment were examined. A single bacterial species was found in 83% of positive cultures. Predominant pathogens were Haemophilus influenzae and Streptococcus pneumoniae which occurred jointly or separately in 50% of positive cultures. Bacteria traditionally considered as non-pathogenic for the lower respiratory tract also appeared to play an aetiological role. Enterobacteriaceae and anaerobes were infrequent. No bacterial growth was found in 11 cases. | [
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PMID:25847 | Antihypertensive effect of trimepranol, a new beta-blocking agent. | Trimepranol (TMP) is a new propranolol-like, non-selective beta-adrenoreceptor blocking drug. Its antihypertensive effect versus placebo was evaluated in a double-blind corss-over study in 25 ambulatory patients, whose supine diastolic blood pressure (BP) was at least 95 mm Hg. After four weeks treatment with placebo, the dose of TMP was titrated weekly until the supine diastolic BP was below 95 mm Hg or intolerable side effects occurred. The trimepranol dose thus determined was 10 mg bid for three patients, 20 mg bid for twelve patients and 40 mg bid for ten patients. The subsequent double-blind cross-over study consisted of two six weeks treatment periods, either with trimepranol followed by placebo (Group I) or in reverse order (Group II). BP and heart rate at the end of these periods were compared. Supine BP fell from 156 +/- 3/105 +/- 2 mm Hg at the end of placebo periods to 140 +/- 2/93 +/- 1 mm Hg (p less than 0.001) for systolic and diastolic BP at the end of trimepranol periods, when the data of Groups I and II are pooled. In 19 out of 25 patients, supine diastolic BP declined below 95 mmHg during the trimepranol period. A statistically significant correlation was found between the antihypertensive and bradycardic effects of trimepranol. Mild side effects occurred in the heart volumes of the patients. We conclude that bid trimepranol is an effective antihypertensive agent. | Antihypertensive effect of trimepranol, a new beta-blocking agent. Trimepranol (TMP) is a new propranolol-like, non-selective beta-adrenoreceptor blocking drug. Its antihypertensive effect versus placebo was evaluated in a double-blind corss-over study in 25 ambulatory patients, whose supine diastolic blood pressure (BP) was at least 95 mm Hg. After four weeks treatment with placebo, the dose of TMP was titrated weekly until the supine diastolic BP was below 95 mm Hg or intolerable side effects occurred. The trimepranol dose thus determined was 10 mg bid for three patients, 20 mg bid for twelve patients and 40 mg bid for ten patients. The subsequent double-blind cross-over study consisted of two six weeks treatment periods, either with trimepranol followed by placebo (Group I) or in reverse order (Group II). BP and heart rate at the end of these periods were compared. Supine BP fell from 156 +/- 3/105 +/- 2 mm Hg at the end of placebo periods to 140 +/- 2/93 +/- 1 mm Hg (p less than 0.001) for systolic and diastolic BP at the end of trimepranol periods, when the data of Groups I and II are pooled. In 19 out of 25 patients, supine diastolic BP declined below 95 mmHg during the trimepranol period. A statistically significant correlation was found between the antihypertensive and bradycardic effects of trimepranol. Mild side effects occurred in the heart volumes of the patients. We conclude that bid trimepranol is an effective antihypertensive agent. | [
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PMID:25851 | Ultrafiltration evidence of ion binding by macromolecules in urine. | In an effort to rationalize the difference between computed estimates of urinary calcium oxalate supersaturation and estimates obtained by equilibrating urine with solid calcium oxalate, we examined ultrasfiltered urine for the binding of urinary ions. Urine was passed through a filter with a nominal passage cutoff of 1000 Daltons. The average reduction in concentration of the ultrafiltrate was 12.7 per cent for oxalate, 11.9 per cent for phosphate, 9.4 percent for calcium, 7.6 per cent for magnesium, and 6.9 per cent for sodium. From these results we conclude that calculations of urinary ion equilibrium will give better estimates if the composition of ultrafiltered urine is used. | Ultrafiltration evidence of ion binding by macromolecules in urine. In an effort to rationalize the difference between computed estimates of urinary calcium oxalate supersaturation and estimates obtained by equilibrating urine with solid calcium oxalate, we examined ultrasfiltered urine for the binding of urinary ions. Urine was passed through a filter with a nominal passage cutoff of 1000 Daltons. The average reduction in concentration of the ultrafiltrate was 12.7 per cent for oxalate, 11.9 per cent for phosphate, 9.4 percent for calcium, 7.6 per cent for magnesium, and 6.9 per cent for sodium. From these results we conclude that calculations of urinary ion equilibrium will give better estimates if the composition of ultrafiltered urine is used. | [
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PMID:25848 | A behavioral approach to post-catastrophic illness work phobias. | Two patients developed work phobias following major catastrophic physical illness. A combined approach to addressing their specific fears of returning to premorbid levels of work function utilized deep muscle relaxation, imagery based desensitization, social reinforcement, and shaping techniques. A significant increase in adaptive function and decrease in depressive symptomatology resulted in both cases within six weeks after initiation of treatment. | A behavioral approach to post-catastrophic illness work phobias. Two patients developed work phobias following major catastrophic physical illness. A combined approach to addressing their specific fears of returning to premorbid levels of work function utilized deep muscle relaxation, imagery based desensitization, social reinforcement, and shaping techniques. A significant increase in adaptive function and decrease in depressive symptomatology resulted in both cases within six weeks after initiation of treatment. | [
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PMID:25852 | Oxalate transport by the human small intestine. | Oxalate was transported in vitro by intestinal brush border cells from rabbit and human by a non-energy-dependent diffusion mechanism. An oxalate-binding protein that had an approximate molecular weight of 73,000 was identified in the human intestine (ileum), was localized in the cytosol cell fraction, and was affected by calcium and magnesium, which increased the oxalate binding. | Oxalate transport by the human small intestine. Oxalate was transported in vitro by intestinal brush border cells from rabbit and human by a non-energy-dependent diffusion mechanism. An oxalate-binding protein that had an approximate molecular weight of 73,000 was identified in the human intestine (ileum), was localized in the cytosol cell fraction, and was affected by calcium and magnesium, which increased the oxalate binding. | [
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PMID:25856 | A quantitative cytochemical method for measuring carbonic anhydrase activity. | Components of a histochemical method for demonstrating carbonic anhydrase activity have been investigated quantitatively. It was found that it is not necessary to use free-floating sections provided the reaction is done in a reaction medium of controlled depth. This permits the use of normal cryostat sections on glass slides, so making this technique applicable to the cytochemical bioassay of gastrin. The better control of the pH of the reaction, and changes in the concentration of phosphate and of cobalt, have resulted in a quantitatively reproducible reaction in the parietal cells of guinea-pig fundus. The reaction product is measured by microdensitometry. The specificity of the carbonic anhydrase reaction has been tested by the response elicited by gastrin acting on the parietal cells in vitro and by the use of acetazolamide. | A quantitative cytochemical method for measuring carbonic anhydrase activity. Components of a histochemical method for demonstrating carbonic anhydrase activity have been investigated quantitatively. It was found that it is not necessary to use free-floating sections provided the reaction is done in a reaction medium of controlled depth. This permits the use of normal cryostat sections on glass slides, so making this technique applicable to the cytochemical bioassay of gastrin. The better control of the pH of the reaction, and changes in the concentration of phosphate and of cobalt, have resulted in a quantitatively reproducible reaction in the parietal cells of guinea-pig fundus. The reaction product is measured by microdensitometry. The specificity of the carbonic anhydrase reaction has been tested by the response elicited by gastrin acting on the parietal cells in vitro and by the use of acetazolamide. | [
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PMID:25859 | Anemia as a stimulus to aortic and carotid chemoreceptors in the cat. | In cats anesthetized with alpha-chloralose, the activities of aortic and carotid chemoreceptor nerves were measured during a control period and during anemia where the hematocrit was lowered in steps by dextran-for-blood exchange. With anemia there was a sustained nonlinear increase in firing of aortic chemoreceptors. There was a greater firing of aortic chemoreceptors for a given lowering of hematocrit from an initial low blood hematocrit than for a similar decrease in hematocrit from an initial high blood hematocrit. Tonic carotid chemoreceptor firing was independent of blood hematocrit and was only transiently increased at the time of dextran-for-blood exchange. The lack of effect of anemia on carotid chemoreceptor activity appeared to be due to sympathetic nerve activity. Section of the sympathetic nerves to the carotid chemoreceptors resulted in an increase in carotid chemoreceptor afferent activity during anemia in a manner similar to the increase in aortic chemoreceptor activity. | Anemia as a stimulus to aortic and carotid chemoreceptors in the cat. In cats anesthetized with alpha-chloralose, the activities of aortic and carotid chemoreceptor nerves were measured during a control period and during anemia where the hematocrit was lowered in steps by dextran-for-blood exchange. With anemia there was a sustained nonlinear increase in firing of aortic chemoreceptors. There was a greater firing of aortic chemoreceptors for a given lowering of hematocrit from an initial low blood hematocrit than for a similar decrease in hematocrit from an initial high blood hematocrit. Tonic carotid chemoreceptor firing was independent of blood hematocrit and was only transiently increased at the time of dextran-for-blood exchange. The lack of effect of anemia on carotid chemoreceptor activity appeared to be due to sympathetic nerve activity. Section of the sympathetic nerves to the carotid chemoreceptors resulted in an increase in carotid chemoreceptor afferent activity during anemia in a manner similar to the increase in aortic chemoreceptor activity. | [
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PMID:25860 | Analysis of postcapillary pH changes in blood in vivo after gas exchange. | A quantitative description of the reaction and transport processes that take place in blood during and after gas exchange in capillaries is developed and used to interpret recently reported experimental results. Included in the computation are 1) CO2-H2CO3 hydration-dehydration reactions in plasma and erythrocytes, 2) CO2 reactions with hemoglobin, 3) O2 binding to hemoglobin, 4) buffering of H+ intra- and extracellularly, 5) HCO3- Cl- exchange across the red cell membrane, 6) diffusion of gases between alveolar gas and blood, and 7) transcellular movement of water. Ion and water fluxes are described assuming passive diffusion down their electrochemical potential gradients. Recent data on the magnitude of the Bohr and Haldane shifts and on carbamate formation in the presence of 2,3-diphosphoglycerate are used. The analysis is used to examine the direction, magnitude, and time course of plasma pH changes in blood leaving the pulmonary capillaries and is shown to preduct results that agree very closely with recently reported experimental measurements in vivo. The time computed for plasma pH equilibration after gas exchange when carbonic anhydrase activity is absent from plasma is so great that blood may never be in complete electrochemical equilibrium as it travels around the circulation in normal man. | Analysis of postcapillary pH changes in blood in vivo after gas exchange. A quantitative description of the reaction and transport processes that take place in blood during and after gas exchange in capillaries is developed and used to interpret recently reported experimental results. Included in the computation are 1) CO2-H2CO3 hydration-dehydration reactions in plasma and erythrocytes, 2) CO2 reactions with hemoglobin, 3) O2 binding to hemoglobin, 4) buffering of H+ intra- and extracellularly, 5) HCO3- Cl- exchange across the red cell membrane, 6) diffusion of gases between alveolar gas and blood, and 7) transcellular movement of water. Ion and water fluxes are described assuming passive diffusion down their electrochemical potential gradients. Recent data on the magnitude of the Bohr and Haldane shifts and on carbamate formation in the presence of 2,3-diphosphoglycerate are used. The analysis is used to examine the direction, magnitude, and time course of plasma pH changes in blood leaving the pulmonary capillaries and is shown to preduct results that agree very closely with recently reported experimental measurements in vivo. The time computed for plasma pH equilibration after gas exchange when carbonic anhydrase activity is absent from plasma is so great that blood may never be in complete electrochemical equilibrium as it travels around the circulation in normal man. | [
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PMID:25875 | Neutral cleanup procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin residues in bovine fat and milk. | A neutral cleanup method for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in milk and animal tissue was developed involving solvent extraction and liquid adsorption chromatography on magnesia-Celite 545, alumina, and Florisil. Cleaned up extracts were subjected to dual-ion analysis in a direct probe high resolution mass spectrometer, interfaced to a multi-channel analyzer for signal averaging. Calibration experiments were carried out with bovine milk and beef fat samples containing added TCDD. The 37CI isotopic isomer of TCDD was added as an internal standard. The response was linear for concentrations in the ppt range, with recoveries about 80%. Milk from a cow fed TCDD was cleaned up by the neutral procedure or, alternatively, a base-acid extraction procedure. The TCDD recoveries for both procedures were essentially the same. Recoveries of TCDD from liver samples of a rat given 14C-TCDD intraperitoneally, subjected to neutral cleanup and radioactive counting, were about 70%. | Neutral cleanup procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin residues in bovine fat and milk. A neutral cleanup method for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in milk and animal tissue was developed involving solvent extraction and liquid adsorption chromatography on magnesia-Celite 545, alumina, and Florisil. Cleaned up extracts were subjected to dual-ion analysis in a direct probe high resolution mass spectrometer, interfaced to a multi-channel analyzer for signal averaging. Calibration experiments were carried out with bovine milk and beef fat samples containing added TCDD. The 37CI isotopic isomer of TCDD was added as an internal standard. The response was linear for concentrations in the ppt range, with recoveries about 80%. Milk from a cow fed TCDD was cleaned up by the neutral procedure or, alternatively, a base-acid extraction procedure. The TCDD recoveries for both procedures were essentially the same. Recoveries of TCDD from liver samples of a rat given 14C-TCDD intraperitoneally, subjected to neutral cleanup and radioactive counting, were about 70%. | [
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PMID:25876 | Atomic absorption spectroscopic determination of lead extracted from acid-solubilized tissues. | A method is presented for determining lead in a variety of tissues. Lyophilized samples are solubilized with nitric acid at room temperature in glass screw-cap culture tubes. Following neutralization with sodium hydroxide and sodium bicarbonate, the lead is extracted into methyl isobutyl ketone as the pyrrolidine dithiocarbamate complex and analyzed by flame atomic absorption spectrophotometry. Brain, heart, liver, lung, and spleen gave recoveries ranging from 92 to 102% with standard deviations of less than 8%. Aorta, kidney, and rib were unsuitable for analysis by this method. A large number of samples can be analyzed without specialized equipment or intricate experimental steps. The detection limit is 35 ng/g tissue (wet weight) and sensitivity is approximately 140 ng/g tissue (wet weight). | Atomic absorption spectroscopic determination of lead extracted from acid-solubilized tissues. A method is presented for determining lead in a variety of tissues. Lyophilized samples are solubilized with nitric acid at room temperature in glass screw-cap culture tubes. Following neutralization with sodium hydroxide and sodium bicarbonate, the lead is extracted into methyl isobutyl ketone as the pyrrolidine dithiocarbamate complex and analyzed by flame atomic absorption spectrophotometry. Brain, heart, liver, lung, and spleen gave recoveries ranging from 92 to 102% with standard deviations of less than 8%. Aorta, kidney, and rib were unsuitable for analysis by this method. A large number of samples can be analyzed without specialized equipment or intricate experimental steps. The detection limit is 35 ng/g tissue (wet weight) and sensitivity is approximately 140 ng/g tissue (wet weight). | [
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PMID:25877 | pH-induced difference spectrophotometric assay of acetazolamide, hydrochlorothiazide, and furosemide. | On the basis of pH-induced spectral changes for acetazolamide, hydrochlorothiazide, and furosemide, 3 different spectrophotometric methods are described. The methods were successfully applied to the assay of these drugs in different pharmaceutical formulations (tablets and ampoules). The accuracy of the analysis is increased by using the suggested methods as compared with conventional spectrophotometry. | pH-induced difference spectrophotometric assay of acetazolamide, hydrochlorothiazide, and furosemide. On the basis of pH-induced spectral changes for acetazolamide, hydrochlorothiazide, and furosemide, 3 different spectrophotometric methods are described. The methods were successfully applied to the assay of these drugs in different pharmaceutical formulations (tablets and ampoules). The accuracy of the analysis is increased by using the suggested methods as compared with conventional spectrophotometry. | [
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PMID:25878 | Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis. | A membrane-bound insoluble alkaline phosphatase (APase) and an extracellular soluble APase were purified, respectively, from a membrane preparation of Bacillus subtilis 6160-BC6, which carries a mutation to produce APase constitutively, and from a culture fluid of a mutant strain. RAN 1, isolated from strain 6160-BC6, which produces an extracellular soluble APase. The two preparations were homogeneous, as judged by sodium dodecyl sulfate discontinuous gel electrophoresis and by gel electrophoreses in the presence of 8 M urea at pH 9.3 and 4.3. RAN 1 APase was crystallized. Both preparations possessed phosphatase and phosphodiesterase activities, and their pH optima were both at 9.5. They were competitively inhibited by phosphate or arsenate and were activated by the addition of Ca2+ but not by Zn2+. The APase and alkaline phosphodiesterase activities seemed to be contained in the same protein molecule. The molecular weight of 6160-BC6 APase was estimated to be 46,000 +/- 1,000, and that of RAN 1 APase was estimated to be 45,000 +/- 1,000. The largest difference between the 6160-BC6 and RAN 1 APase's was in solubility in low-ionic-strength solutions. Present results suggest that each enzyme is composed of a single polypeptide chain and that 6160-BC6 APase aggregates in solutions of low ionic strength. | Purification and characterization of extracellular soluble and membrane-bound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis. A membrane-bound insoluble alkaline phosphatase (APase) and an extracellular soluble APase were purified, respectively, from a membrane preparation of Bacillus subtilis 6160-BC6, which carries a mutation to produce APase constitutively, and from a culture fluid of a mutant strain. RAN 1, isolated from strain 6160-BC6, which produces an extracellular soluble APase. The two preparations were homogeneous, as judged by sodium dodecyl sulfate discontinuous gel electrophoresis and by gel electrophoreses in the presence of 8 M urea at pH 9.3 and 4.3. RAN 1 APase was crystallized. Both preparations possessed phosphatase and phosphodiesterase activities, and their pH optima were both at 9.5. They were competitively inhibited by phosphate or arsenate and were activated by the addition of Ca2+ but not by Zn2+. The APase and alkaline phosphodiesterase activities seemed to be contained in the same protein molecule. The molecular weight of 6160-BC6 APase was estimated to be 46,000 +/- 1,000, and that of RAN 1 APase was estimated to be 45,000 +/- 1,000. The largest difference between the 6160-BC6 and RAN 1 APase's was in solubility in low-ionic-strength solutions. Present results suggest that each enzyme is composed of a single polypeptide chain and that 6160-BC6 APase aggregates in solutions of low ionic strength. | [
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PMID:25879 | Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli. | Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation. | Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli. Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation. | [
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PMID:25880 | Assimilatory sulfate reduction in an Escherichia coli mutant lacking thioredoxin activity. | An investigation of sulfate reduction in B tsnC*7004, a mutant of Escherichia coli lacking thioredoxin, is reported. Although thioredoxin is indispensable for the adenosine 3'-phosphate 5'-phosphosulfate (PAPS) sulfotransferase reaction under the usual conditions of assay in extracts of wild-type cells, the mutant grew as well as the wild type on sulfate, indicating that sulfate reduction is not rate limiting for growth. Another cofactor for the PAPS sulfotransferase reaction was found in extracts of the mutant that is absent from wild type cells. This cofactor was indistinguishable from thioredoxin in molecular weight but had a slightly different isoelectric point, allowing a separation of the two types of molecules by isoelectric focusing. Whereas electrons from nicotinamide adenine dinucleotide phosphate, reduced form, could be transferred via thioredoxin reductase or via glutathione and glutathione reductase to reduce thioredoxin in extracts of wild-type cells, electrons from nicotinamide adenine dinucleotide, reduced form, could only be transferred to the cofactor of the mutant via glutathione and glutathione reductase. All of the other available mutants blocked in sulfate reduction in E. coli contained normal levels of thioredoxin. The "PAPS reductase" mutant is shown to be blocked in the PAPS sulfotransferase reaction. We conclude that the cofactor found in mutant B tsnC*7004 is probably a mutated thioredoxin with an amino acid substitution that alters the isoelectric point and the reactivity with thioredoxin reductase. | Assimilatory sulfate reduction in an Escherichia coli mutant lacking thioredoxin activity. An investigation of sulfate reduction in B tsnC*7004, a mutant of Escherichia coli lacking thioredoxin, is reported. Although thioredoxin is indispensable for the adenosine 3'-phosphate 5'-phosphosulfate (PAPS) sulfotransferase reaction under the usual conditions of assay in extracts of wild-type cells, the mutant grew as well as the wild type on sulfate, indicating that sulfate reduction is not rate limiting for growth. Another cofactor for the PAPS sulfotransferase reaction was found in extracts of the mutant that is absent from wild type cells. This cofactor was indistinguishable from thioredoxin in molecular weight but had a slightly different isoelectric point, allowing a separation of the two types of molecules by isoelectric focusing. Whereas electrons from nicotinamide adenine dinucleotide phosphate, reduced form, could be transferred via thioredoxin reductase or via glutathione and glutathione reductase to reduce thioredoxin in extracts of wild-type cells, electrons from nicotinamide adenine dinucleotide, reduced form, could only be transferred to the cofactor of the mutant via glutathione and glutathione reductase. All of the other available mutants blocked in sulfate reduction in E. coli contained normal levels of thioredoxin. The "PAPS reductase" mutant is shown to be blocked in the PAPS sulfotransferase reaction. We conclude that the cofactor found in mutant B tsnC*7004 is probably a mutated thioredoxin with an amino acid substitution that alters the isoelectric point and the reactivity with thioredoxin reductase. | [
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PMID:25881 | Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes. | Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells. | Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes. Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells. | [
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PMID:25882 | Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. | Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport. | Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport. | [
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PMID:25883 | Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad. | Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol. | Purification and characterization of a secondary alcohol dehydrogenase from a pseudomonad. Growth of Pseudomonas sp. NRRL B3266 in the presence of oleic acid resulted in the induction of two enzymes: oleate hydratase, which produced 10(R)hydroxyoctadecanoate, and hydroxyoctadecanoate dehydrogenase, which catalyzed the oxidized nicotinamide adenine dinucleotide-dependent production of 10-oxooctadecanoate. This latter enzyme was purified to homogeneity and shown to consist of two polypeptide chains of about 29,000 daltons each. The enzyme had a broad substrate specificity, catalyzing the dehydrogenation of a number of 18-carbon hydroxy fatty acids. The kinetic parameters for various 10- and 12-hydroxy fatty acids were similar (Km ca. 5 micron and Vmax ca. 50 to 200 mumol/min per mg of protein). The enzyme also catalyzed the dehydrogenation of unsubstituted secondary alcohols. The effectiveness of these alcohols as substrates was highly dependent on their hydrophobicity, the Km decreasing from 9 mM for 4-heptanol to 7 micron for 6-dodecanol. Inhibition of the enzyme by primary alcohols also showed a dependence on hydrophobicity, the Ki decreasing from 350 mM for methanol to 90 micron for decanol. | [
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PMID:25884 | Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci. | Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g. | Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci. Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g. | [
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PMID:25885 | Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties. | A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively. | Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties. A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively. | [
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PMID:25886 | Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization. | The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding. | Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization. The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding. | [
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PMID:25887 | Acetyl-CoA carboxylase. Evidence for polymeric filament to protomer transition in the intact avian liver cell. | Digitonin treatment of chick liver cells in monolayer culture perforates the plasma membrane, causing release of acetyl-CoA carboxylase and other cytosolic enzymes. The rate of carboxylase release is affected by conditions known to alter the position of the protomer-polymer (filament) equilibrium of the enzyme. Citrate, an allosteric activator of the carboxylase, induces polymerization of the protomeric avidin-sensitive form giving rise to the avidin-insensitive polymeric filamentous form. When cells are exposed to N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate which lowers intracellular citrate levels, the rate of carboxylase release from digitonin-treated cells is greatly accelerated. The presence of avidin, which rapidly enters the cell during digitonin treatment, inactivates carboxylase under conditions that promote depolymerization and rapid release, but not under conditions which promote polymerization and slow release. These findings indicate that carboxylase filaments exist in the intact chick liver cell when the cytoplasmic citrate level is high and undergo depolymerization when citrate levels fall. | Acetyl-CoA carboxylase. Evidence for polymeric filament to protomer transition in the intact avian liver cell. Digitonin treatment of chick liver cells in monolayer culture perforates the plasma membrane, causing release of acetyl-CoA carboxylase and other cytosolic enzymes. The rate of carboxylase release is affected by conditions known to alter the position of the protomer-polymer (filament) equilibrium of the enzyme. Citrate, an allosteric activator of the carboxylase, induces polymerization of the protomeric avidin-sensitive form giving rise to the avidin-insensitive polymeric filamentous form. When cells are exposed to N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate which lowers intracellular citrate levels, the rate of carboxylase release from digitonin-treated cells is greatly accelerated. The presence of avidin, which rapidly enters the cell during digitonin treatment, inactivates carboxylase under conditions that promote depolymerization and rapid release, but not under conditions which promote polymerization and slow release. These findings indicate that carboxylase filaments exist in the intact chick liver cell when the cytoplasmic citrate level is high and undergo depolymerization when citrate levels fall. | [
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PMID:25889 | Hemoglobins of the killifish Fundulus heteroclitus. Separation, characterization, and a model for the subunit compositions. | Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model. | Hemoglobins of the killifish Fundulus heteroclitus. Separation, characterization, and a model for the subunit compositions. Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model. | [
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PMID:25890 | Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin. | The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations. | Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin. The hydrogen exchange kinetics of human oxy-, deoxy-, and cyanomethemoglobin have been measured as a function of pH by the tritium tracer method. At 5 degrees C and in phosphate buffer both liganded and unliganded forms of ferrohemoglobin exhibit deviations from the regular pH dependence of exchange that is characteristic of cyanomethemoglobin. In oxyhemoglobin, the deviation from the normal exchange pattern is centered at pH 7.4 and is in the direction of increased exchange or solvent accessibility. The effect in deoxyhemogloin, while occurring at the same pH and being of the same order of magnitude, is in the opposite direction, thus suggesting a pH-induced conformational transition leading to a less accessible structure. The width of these pH-induced deviations in solvent accessibility is approximately 1 pH unit in both cases. We propose a model in which specific interactions between charged groups in both froms of ferrohemoglobin account for these deviations. | [
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PMID:25891 | Enzymatic methylation of carboxyl groups of chromaffin granule membrane proteins. | Carboxyl groups of membrane and soluble proteins from bovine adrenal medulla chromaffin granules were enzymatically methylated. The methylated peptides were resolved using gel electrophoresis under acidic conditions in the presence of N-cetylpyridinium chloride. There was a selective methylation of two groups of membrane peptides which did not correspond to any of the chromaffin granule soluble proteins. Dopamine beta-hydroxylase, an acidic protein accounting for up to 25% of the membrane proteins, was a poor substrate for protein carboxylmethylase. The methyl esters of membrane proteins were more labile than those of the chromaffin granule soluble proteins. At all pH values tested, membrane protein-methyl esters were hydrolyzed three times more rapidly than the soluble protein-methyl esters. | Enzymatic methylation of carboxyl groups of chromaffin granule membrane proteins. Carboxyl groups of membrane and soluble proteins from bovine adrenal medulla chromaffin granules were enzymatically methylated. The methylated peptides were resolved using gel electrophoresis under acidic conditions in the presence of N-cetylpyridinium chloride. There was a selective methylation of two groups of membrane peptides which did not correspond to any of the chromaffin granule soluble proteins. Dopamine beta-hydroxylase, an acidic protein accounting for up to 25% of the membrane proteins, was a poor substrate for protein carboxylmethylase. The methyl esters of membrane proteins were more labile than those of the chromaffin granule soluble proteins. At all pH values tested, membrane protein-methyl esters were hydrolyzed three times more rapidly than the soluble protein-methyl esters. | [
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PMID:25894 | Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts. | An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals. | Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts. An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals. | [
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PMID:25896 | Isolation and characterization of ornithine transcarbamylase from normal human liver. | We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue. | Isolation and characterization of ornithine transcarbamylase from normal human liver. We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue. | [
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PMID:25898 | Changes in hepatic levels of tyrosine aminotransferase messenger RNA during induction by hydrocortisone. | Messenger RNA specific for tyrosine aminotransferase was quantitated by microinjection into oocytes of Xenopus laevis. The heterologously translated enzyme was identified by specific immunoprecipitation and found to be identical with authentic aminotransferase by several criteria. The level of functional message present in rat liver increases during hydrocortisone induction, and this increase is directly proportional to the increased rate of synthesis of the enzyme. Kinetic analysis of the changes in tyrosine aminotransferase mRNA levels during induction and withdrawal indicates that the steroid does not affect the stability of the message, which has a half-life of approximately 1.2 h. Hydrocortisone, therefore, acts to increase the rate of synthesis of the specific messenger by stimulating either its transcription or processing to functional mRNA. | Changes in hepatic levels of tyrosine aminotransferase messenger RNA during induction by hydrocortisone. Messenger RNA specific for tyrosine aminotransferase was quantitated by microinjection into oocytes of Xenopus laevis. The heterologously translated enzyme was identified by specific immunoprecipitation and found to be identical with authentic aminotransferase by several criteria. The level of functional message present in rat liver increases during hydrocortisone induction, and this increase is directly proportional to the increased rate of synthesis of the enzyme. Kinetic analysis of the changes in tyrosine aminotransferase mRNA levels during induction and withdrawal indicates that the steroid does not affect the stability of the message, which has a half-life of approximately 1.2 h. Hydrocortisone, therefore, acts to increase the rate of synthesis of the specific messenger by stimulating either its transcription or processing to functional mRNA. | [
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PMID:25900 | Magnesium-induced inner membrane aggregation in heart mitochondria. | Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge. | Magnesium-induced inner membrane aggregation in heart mitochondria. Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge. | [
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PMID:25901 | Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. | When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize. | Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize. | [
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PMID:25902 | Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm. | Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding inophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the ammount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomer. | Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm. Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding inophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaCl at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the ammount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomer. | [
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PMID:25903 | The development of lysosomal apparatus. I. Lysosomal enzyme activities in the liver of mice at perinatal stages and those of their mothers. | The enzymatic activity of five acid hydrolases: acid phosphatase, arylsulfatase A, deoxyribonuclease, beta-glucuronidase, and cathepsin D, was assayed in fetal (fifteenth and eighteenth days of pregnancy) and neonatal (Days 0, 5, 10, and 15 post-partum) mouse liver. With the exception of cathepsin D, the activity increased around birth to levels varying according to the enzyme. Histochemical observations of other authors appear to justify, at least in part, the present results, which indicate that late days of fetal development and early neonatal life may constitute a transitional stage to full lysosomal enzyme functionality of the adult organ. The livers of the mothers were also assayed for the same enzymes. Each activity showed a peculiar pattern which was, in turn, different from that found in the liver of the litter for the same enzyme, probably as a cause of the metabolic requirement of the gland. The hypothesis that the lysosomes are heterogeneous in their enzyme composition is suggested by the variety of enzymatic patterns found in the liver of the litters and their mothers. | The development of lysosomal apparatus. I. Lysosomal enzyme activities in the liver of mice at perinatal stages and those of their mothers. The enzymatic activity of five acid hydrolases: acid phosphatase, arylsulfatase A, deoxyribonuclease, beta-glucuronidase, and cathepsin D, was assayed in fetal (fifteenth and eighteenth days of pregnancy) and neonatal (Days 0, 5, 10, and 15 post-partum) mouse liver. With the exception of cathepsin D, the activity increased around birth to levels varying according to the enzyme. Histochemical observations of other authors appear to justify, at least in part, the present results, which indicate that late days of fetal development and early neonatal life may constitute a transitional stage to full lysosomal enzyme functionality of the adult organ. The livers of the mothers were also assayed for the same enzymes. Each activity showed a peculiar pattern which was, in turn, different from that found in the liver of the litter for the same enzyme, probably as a cause of the metabolic requirement of the gland. The hypothesis that the lysosomes are heterogeneous in their enzyme composition is suggested by the variety of enzymatic patterns found in the liver of the litters and their mothers. | [
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PMID:25904 | PH oscillations in cell suspensions of Dictyostelium discoideum: their relation to cyclic-amp signals. | Cells of Dictyostelium discoideum known to release cyclic AMP (cAMP) rhythmically in the form of pulses, change with the same period of about 8 min the pH of their medium. The pH is used here as an indicator to investigate the effect of externally added cAMP pulses on the oscillations. Both a temporary increase in amplitude and a permanent phase shift can be induced. The phase-response curve indicates that the period can be increased and decreased by rhythmic stimulation with cAMP pulses. | PH oscillations in cell suspensions of Dictyostelium discoideum: their relation to cyclic-amp signals. Cells of Dictyostelium discoideum known to release cyclic AMP (cAMP) rhythmically in the form of pulses, change with the same period of about 8 min the pH of their medium. The pH is used here as an indicator to investigate the effect of externally added cAMP pulses on the oscillations. Both a temporary increase in amplitude and a permanent phase shift can be induced. The phase-response curve indicates that the period can be increased and decreased by rhythmic stimulation with cAMP pulses. | [
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PMID:25909 | Interpatient microbiological cross-contamination after dental radiographic examination. | Pairs of patients were evaluated for microbiological cross-contamination after radiographic examination. In 30 of these pairs of patients there was the possibility of transference of S pyogenes, S aureus, or D pneumoniae. Such transference was observed in 23 (77%) of these 30 pairs of patients. The vectors for such transfer include the hands of the X-ray technician and the radiographic equipment. Further, it was found that each of these organisms would survive for at least 48 hours after being placed on an X-ray tube. Since interpatient microbiological cross-contamination can occur after routine radiographic examination, in some cases, disinfection of the radiographic equipment is indicated. | Interpatient microbiological cross-contamination after dental radiographic examination. Pairs of patients were evaluated for microbiological cross-contamination after radiographic examination. In 30 of these pairs of patients there was the possibility of transference of S pyogenes, S aureus, or D pneumoniae. Such transference was observed in 23 (77%) of these 30 pairs of patients. The vectors for such transfer include the hands of the X-ray technician and the radiographic equipment. Further, it was found that each of these organisms would survive for at least 48 hours after being placed on an X-ray tube. Since interpatient microbiological cross-contamination can occur after routine radiographic examination, in some cases, disinfection of the radiographic equipment is indicated. | [
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PMID:25910 | Tear pH: how predictable? | While the tear pH of a patient is now known to be continually shifting throughout the waking day, such variations were found to be contained within fairly narrow limits (a range here of about 0.6 of a pH unit). Further, sufficient repetition of these shifts occurred over 10 days observed that patterns distinctive of the individual patient often emerged. | Tear pH: how predictable? While the tear pH of a patient is now known to be continually shifting throughout the waking day, such variations were found to be contained within fairly narrow limits (a range here of about 0.6 of a pH unit). Further, sufficient repetition of these shifts occurred over 10 days observed that patterns distinctive of the individual patient often emerged. | [
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PMID:25934 | A novel immunoadsorbent: use for the preparation of monospecific antibodies to the hepatitis B antigen. | Controlled pore glass (CPG) adsorbs hepatitis B surface antigen (HBsAg) from whole plasma with a high degree of specificity. The resultant complex is stable at acid pH and in the presence of high concentrations of sodium thiocyanate. The adsorbed HBsAg is qualitatively and quantitatively similar to the soluble material in its ability to bind antibodies to HBsAg (anti-HBs). The HBsAg in 1 ml of strongly reactive plasma is adsorbed by 100 mg of CPG, which can then specifically bind 32,000 passive hemagglutination units of anti-HBs. Bound antibody can be eluted in 77% yield by acid or by chaotropic ions and the CPG-HBsAg complex can be reused in further adsorption-elution cycles. Antibody to HBsAg can be purified 144-fold in a single step by using this technique. The preparation of monospecific subtyping reagents for HBsAg and of immunochemically purified anti-HBs is described. | A novel immunoadsorbent: use for the preparation of monospecific antibodies to the hepatitis B antigen. Controlled pore glass (CPG) adsorbs hepatitis B surface antigen (HBsAg) from whole plasma with a high degree of specificity. The resultant complex is stable at acid pH and in the presence of high concentrations of sodium thiocyanate. The adsorbed HBsAg is qualitatively and quantitatively similar to the soluble material in its ability to bind antibodies to HBsAg (anti-HBs). The HBsAg in 1 ml of strongly reactive plasma is adsorbed by 100 mg of CPG, which can then specifically bind 32,000 passive hemagglutination units of anti-HBs. Bound antibody can be eluted in 77% yield by acid or by chaotropic ions and the CPG-HBsAg complex can be reused in further adsorption-elution cycles. Antibody to HBsAg can be purified 144-fold in a single step by using this technique. The preparation of monospecific subtyping reagents for HBsAg and of immunochemically purified anti-HBs is described. | [
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PMID:25935 | Sphingomyelinase in pig and human epidermis. | The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine. | Sphingomyelinase in pig and human epidermis. The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine. | [
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PMID:25938 | Collecting duct hydrogen ion secretion in the rabbit: role of potassium. | The purpose of this study was to investigate distal nephron hydrogen ion secretion in the intact animal. The rabbit was chosen as the experimental model because it produces acid urine containing little ammonium. Upon replacement of the usual rabbit diet with milk, plus administration of an acid load (10 mEq/kg), the urine pH fell consistently from very alkaline values (PH greater than 7.4) to 4.8 +/- 0.2. Despite the ability to achieve high urine-to-blood hydrogen ion concentration gradients, the U-B PCO2, an index of collecting duct hydrogen ion secretion, was virtually zero. In these studies, the urine bicarbonate and buffer concentration were comparable to those observed in dog, rat, and man in which a high U-B PCO2 gradient was achieved. The rabbits studied had low plasma potassium concentrations (less than 3 mEq/L). Since potassium deficiency has been implicated in impaired urine acidification, potassium was administered, and it resulted in an increase in collecting duct hydrogen ion secretion as evidenced by a further fall in minimum urine pH during acidemia and a prompt rise in the U-B PCO2 during alkali administration. In summary, rabbits had a very low but not absent rate of collecting duct hydrogen ion secretion. Potassium administration increased the rate of hydrogen ion secretion in a segment of the collecting duct in which hydrogen ion secretion is reflected by an increase U-B PCO2. | Collecting duct hydrogen ion secretion in the rabbit: role of potassium. The purpose of this study was to investigate distal nephron hydrogen ion secretion in the intact animal. The rabbit was chosen as the experimental model because it produces acid urine containing little ammonium. Upon replacement of the usual rabbit diet with milk, plus administration of an acid load (10 mEq/kg), the urine pH fell consistently from very alkaline values (PH greater than 7.4) to 4.8 +/- 0.2. Despite the ability to achieve high urine-to-blood hydrogen ion concentration gradients, the U-B PCO2, an index of collecting duct hydrogen ion secretion, was virtually zero. In these studies, the urine bicarbonate and buffer concentration were comparable to those observed in dog, rat, and man in which a high U-B PCO2 gradient was achieved. The rabbits studied had low plasma potassium concentrations (less than 3 mEq/L). Since potassium deficiency has been implicated in impaired urine acidification, potassium was administered, and it resulted in an increase in collecting duct hydrogen ion secretion as evidenced by a further fall in minimum urine pH during acidemia and a prompt rise in the U-B PCO2 during alkali administration. In summary, rabbits had a very low but not absent rate of collecting duct hydrogen ion secretion. Potassium administration increased the rate of hydrogen ion secretion in a segment of the collecting duct in which hydrogen ion secretion is reflected by an increase U-B PCO2. | [
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PMID:25939 | The distribution of bumps in the tail of the locust photoreceptor afterpotential. | An extended tail or prolonged depolarizing afterpotential (PDA) follows the receptor potential of a locust retinula cell when the stimulating light is in the intensity range that saturates the receptor potential. The amplitude and duration of this afterpotential depend on the intensity and duration of the stimulus. As the afterpotential decays, apparently exponentially, it becomes resolved into bumps, which we call light-induced dark bumps (LID bumps). The intervals between light-induced dark bumps are distributed in a way that is indistinguishable from a random (Poisson) distribution. As previously demonstrated, LID bumps are indistinguishable from bumps directly induced by low intensity light in light-adapted cells, which in turn grade into the slightly larger bumps produced, each by a single photon, in dark-adapted cells. The light-induced dark bumps continue for up to an hour in darkness, slowly becoming like dark-adapted bumps in amplitude and shape. To account for the random occurrence and discrete features of bumps after so long a latency, we propose that intense light generates a significant amount of an intermediate molecule or packet which decays slowly to start the same process that normally generates bumps with a short delay. | The distribution of bumps in the tail of the locust photoreceptor afterpotential. An extended tail or prolonged depolarizing afterpotential (PDA) follows the receptor potential of a locust retinula cell when the stimulating light is in the intensity range that saturates the receptor potential. The amplitude and duration of this afterpotential depend on the intensity and duration of the stimulus. As the afterpotential decays, apparently exponentially, it becomes resolved into bumps, which we call light-induced dark bumps (LID bumps). The intervals between light-induced dark bumps are distributed in a way that is indistinguishable from a random (Poisson) distribution. As previously demonstrated, LID bumps are indistinguishable from bumps directly induced by low intensity light in light-adapted cells, which in turn grade into the slightly larger bumps produced, each by a single photon, in dark-adapted cells. The light-induced dark bumps continue for up to an hour in darkness, slowly becoming like dark-adapted bumps in amplitude and shape. To account for the random occurrence and discrete features of bumps after so long a latency, we propose that intense light generates a significant amount of an intermediate molecule or packet which decays slowly to start the same process that normally generates bumps with a short delay. | [
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PMID:25940 | Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. | I. Blood samples were taken during prolonged hypoxia experiments in which the inspired water oxygen tension was less than 10 mmHg. The oxygen tension of the post-branchial blood was about 5 mmHg and its pH shows a significant lowering from normoxic levels. 2. The decrease in blood pH is correlated with increases in levels of lactate and pyruvate. The lactate/pyruvate ratio increases during hypoxia. 3. An increase in blood succinate was also found, and strongly suggests the accumulation of multiple anaerobic end-products within the tissues. 4. Recovery of normoxic levels of succinate takes place almost immediately following the restart of ventilation whereas the decrease in lactate concentration is slower. 5. It is concluded that these adaptations may be related to the habitat of the fish at low tide in pools where the Po2 may fall very markedly. | Some responses of the electric ray (Torpedo marmorata) to low ambient oxygen tensions. I. Blood samples were taken during prolonged hypoxia experiments in which the inspired water oxygen tension was less than 10 mmHg. The oxygen tension of the post-branchial blood was about 5 mmHg and its pH shows a significant lowering from normoxic levels. 2. The decrease in blood pH is correlated with increases in levels of lactate and pyruvate. The lactate/pyruvate ratio increases during hypoxia. 3. An increase in blood succinate was also found, and strongly suggests the accumulation of multiple anaerobic end-products within the tissues. 4. Recovery of normoxic levels of succinate takes place almost immediately following the restart of ventilation whereas the decrease in lactate concentration is slower. 5. It is concluded that these adaptations may be related to the habitat of the fish at low tide in pools where the Po2 may fall very markedly. | [
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PMID:25941 | The identification of an octopaminergic neurone and the modulation of a myogenic rhythm in the locust. | An octopaminergic neurone in an insect is demonstrated. This cell (DUMETi) is the dorsal unpaired median neurone which projects to the extensor tibiae muscle of the locust metathoracic leg. Its soma was physiologically identified, isolated and shown to contain about 0.1 pmol of octopamine. Octopamine is about four times more concentrated in the axon than in the soma. The concentration in the soma is at least 800 times more than that in the soma of an identified motoneurone (which controls the fast extensor of the tibia). The effects of DUMETi on a myogenic rhythm in the extensor muscle of the locust metathoracic leg can be mimicked by superfusion with low concentrations of octopamine. The myogenic bundle possesses at least two types of aminergic receptor: one which slows the rhythm (and has a high-affinity for octopamine) and a second which accelerates the rhythm (and has a low-affinity for octopamine but a high-affinity for the indolalkylamine, 5-hydroxytryptamine). The roles of the two receptor types in modulating the rhythm are discussed in relation to the function of the rhythm. | The identification of an octopaminergic neurone and the modulation of a myogenic rhythm in the locust. An octopaminergic neurone in an insect is demonstrated. This cell (DUMETi) is the dorsal unpaired median neurone which projects to the extensor tibiae muscle of the locust metathoracic leg. Its soma was physiologically identified, isolated and shown to contain about 0.1 pmol of octopamine. Octopamine is about four times more concentrated in the axon than in the soma. The concentration in the soma is at least 800 times more than that in the soma of an identified motoneurone (which controls the fast extensor of the tibia). The effects of DUMETi on a myogenic rhythm in the extensor muscle of the locust metathoracic leg can be mimicked by superfusion with low concentrations of octopamine. The myogenic bundle possesses at least two types of aminergic receptor: one which slows the rhythm (and has a high-affinity for octopamine) and a second which accelerates the rhythm (and has a low-affinity for octopamine but a high-affinity for the indolalkylamine, 5-hydroxytryptamine). The roles of the two receptor types in modulating the rhythm are discussed in relation to the function of the rhythm. | [
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PMID:25942 | Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. | Bone marrow (BM) and skin allografts from C57BL/Ka (H-2b/b) mice were transplanted to BALB/c (H-2d/d) recipients treated with total lymphoid irradiation (TLI), whole-body irradiation (WBI), or fractionated thymic irradiation TLI prolonged skin allograft survival about five times as long as that in untreated controls, and allowed for permanent engraftment of BM cells in approximately equal to 90% of recipients. None of the BM recipients showed clinical signs of graft-versus-host disease (GVHD) (diarrhea, weight loss, hunched back, etc.). On the other hand, recipients given WBI and allogeneic BM cells developed severe clinical GVHD. The majority of the latter recipients died within 12 days after BM transplantation, and 95% died within 61 days. Although TLI protected BALB/c mice against GVHD induced by BM cells, all recipients given TLI and allogeneic spleen cells developed lethal GVHD. Thymic irradiation alone marginally prolonged skin allograft survival, and did not allow for allogeneic BM engraftment. These results suggest that TLI may be a useful regimen in clinical BM transplantation, since this form of radiotherapy is used extensively in humans and has few severe side effects. | Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. Bone marrow (BM) and skin allografts from C57BL/Ka (H-2b/b) mice were transplanted to BALB/c (H-2d/d) recipients treated with total lymphoid irradiation (TLI), whole-body irradiation (WBI), or fractionated thymic irradiation TLI prolonged skin allograft survival about five times as long as that in untreated controls, and allowed for permanent engraftment of BM cells in approximately equal to 90% of recipients. None of the BM recipients showed clinical signs of graft-versus-host disease (GVHD) (diarrhea, weight loss, hunched back, etc.). On the other hand, recipients given WBI and allogeneic BM cells developed severe clinical GVHD. The majority of the latter recipients died within 12 days after BM transplantation, and 95% died within 61 days. Although TLI protected BALB/c mice against GVHD induced by BM cells, all recipients given TLI and allogeneic spleen cells developed lethal GVHD. Thymic irradiation alone marginally prolonged skin allograft survival, and did not allow for allogeneic BM engraftment. These results suggest that TLI may be a useful regimen in clinical BM transplantation, since this form of radiotherapy is used extensively in humans and has few severe side effects. | [
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PMID:25943 | Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging. | Marrow stem cell lines from old donors and those from young controls gave equally rapid rates of colony growth on spleens of irradiated mice. Old and young stem cell lines competed equally well with chromosomally marked marrow stem cells from a young donor in producing cell types that are stimulated by bleeding; old cells competed 70% as well as young in producing cell types stimulated by phytohemagglutinin (PHA) in vitro. After a single serial transplantation, the rates of colony growth declined 1.5- to 2.5-fold, and the ability to compete declined 2- to 4-fold for bleeding-stimulated and 4- to 10-fold for PHA-stimulated cells. Thus, immediate stem cell proliferative capacities decline much more after one serial transplantation than after a lifetime of normal function. | Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging. Marrow stem cell lines from old donors and those from young controls gave equally rapid rates of colony growth on spleens of irradiated mice. Old and young stem cell lines competed equally well with chromosomally marked marrow stem cells from a young donor in producing cell types that are stimulated by bleeding; old cells competed 70% as well as young in producing cell types stimulated by phytohemagglutinin (PHA) in vitro. After a single serial transplantation, the rates of colony growth declined 1.5- to 2.5-fold, and the ability to compete declined 2- to 4-fold for bleeding-stimulated and 4- to 10-fold for PHA-stimulated cells. Thus, immediate stem cell proliferative capacities decline much more after one serial transplantation than after a lifetime of normal function. | [
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PMID:25944 | A comparative study of the electrode systems of three pH and blood gas apparatus. | We present a comparative evaluation of the electrode systems of three modern blood gas analysers: IL-413, ABL-1 and AVL-937C. The response curves, accuracy and precision of the pH-, pCO2- and pO2-electrodes were established with tonometered blood and buffer solutions. pH values (range 6.8-7.8) measured on the AVL deviate (-0.03 pH for blood and +0.03 pH for buffer) from those of BMS2 Mk2; whereas on the IL and ABL analysers the pH values deviate by not more than 0.01 pH. The standard deviation was better than 0.005 pH. pCO2 values of blood and buffer (range 14-106 mm Hg) deviate from the calculated tonometer values by quantities ranging from 3 to 10 mm Hg. The average precision (CV)1) of the pCO2 measurement on each analyser was better than 1.8%. pO2 values of blood (range 0-130 mm Hg) did not differ by more than 3 mm Hg from the calculated values. Above 130 mm Hg a linear negative increasing difference was seen. For buffer solutions a linear relationship between pO2 difference and pO2 value was found over the whole range from zero up to 642 mm Hg: a positive difference below and a negative difference above the pO2 of the previous calibration; if the calibration pO2 is higher, the sample pO2 is shifted to a higher value. The average precision of the pO2 measurements was better than 3%. In the (patho)-physiological range the three instruments may provide suitable results for the clinician. Suggestions are made for standardization and improvement of the electrode systems. | A comparative study of the electrode systems of three pH and blood gas apparatus. We present a comparative evaluation of the electrode systems of three modern blood gas analysers: IL-413, ABL-1 and AVL-937C. The response curves, accuracy and precision of the pH-, pCO2- and pO2-electrodes were established with tonometered blood and buffer solutions. pH values (range 6.8-7.8) measured on the AVL deviate (-0.03 pH for blood and +0.03 pH for buffer) from those of BMS2 Mk2; whereas on the IL and ABL analysers the pH values deviate by not more than 0.01 pH. The standard deviation was better than 0.005 pH. pCO2 values of blood and buffer (range 14-106 mm Hg) deviate from the calculated tonometer values by quantities ranging from 3 to 10 mm Hg. The average precision (CV)1) of the pCO2 measurement on each analyser was better than 1.8%. pO2 values of blood (range 0-130 mm Hg) did not differ by more than 3 mm Hg from the calculated values. Above 130 mm Hg a linear negative increasing difference was seen. For buffer solutions a linear relationship between pO2 difference and pO2 value was found over the whole range from zero up to 642 mm Hg: a positive difference below and a negative difference above the pO2 of the previous calibration; if the calibration pO2 is higher, the sample pO2 is shifted to a higher value. The average precision of the pO2 measurements was better than 3%. In the (patho)-physiological range the three instruments may provide suitable results for the clinician. Suggestions are made for standardization and improvement of the electrode systems. | [
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PMID:25946 | Activation of rat pheochromocytoma tyrosine hydroxylase by a cyclic AMP-dependent protein kinase in a cell-free system. | Short term exposure of PC-12 cells to dibutyryl cyclic AMP (dB-cAMP) results in an activation of tyrosine hydroxylase. In the cell-free system the PC-12 tyrosine hydroxylase activity is stimulated by addition of c-AMP, Mg+2 and ATP. Exogenous c-AMP dependent protein kinase further stumulates tyrosine hydroxylase activity. The kinetic data suggests that the PC-12 tyrosine hydroxylase in the basal state is in a non-phosphorylated form but under phosphorylating conditions the enzyme is activated and its kinetics properties are altered. | Activation of rat pheochromocytoma tyrosine hydroxylase by a cyclic AMP-dependent protein kinase in a cell-free system. Short term exposure of PC-12 cells to dibutyryl cyclic AMP (dB-cAMP) results in an activation of tyrosine hydroxylase. In the cell-free system the PC-12 tyrosine hydroxylase activity is stimulated by addition of c-AMP, Mg+2 and ATP. Exogenous c-AMP dependent protein kinase further stumulates tyrosine hydroxylase activity. The kinetic data suggests that the PC-12 tyrosine hydroxylase in the basal state is in a non-phosphorylated form but under phosphorylating conditions the enzyme is activated and its kinetics properties are altered. | [
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PMID:25945 | Intestinal transport of weak electrolytes: Determinants of influx at the luminal surface. | The determinants of weak electrolyte influx into everted segments of rat small intestine have been studied. Preliminary experiments showed that the observed influxes could be described as unidirectional, diffusional fluxes of the nonionized compound uncomplicated by a parallel ionic component. It is shown that the determinants of weak electrolyte influx in this situation may be described in terms of the resistance of the unstirred layer to movement from the bulk phase to the cell surface, the degree of ionization of the weak electrolyte at the cell surface, and the cellular permeability to the nonionized weak electrolyte. Quantitative considerations indicated that the unstirred layer was totally rate-limiting in the cases of some poorly ionized, or highly permeant compounds, but the unstirred layer was not totally rate limiting for most of the compounds studied. Calculation of cellular permeabilities for the nonionized forms of weak electrolytes required assumptions to be made concerning the pH value in the surface fluid layer. A uniform set of permeability data including both weak acids and weak bases was obtained only when it was assumed that the pH in the surface fluid layer was equal to that in the bulk phase, and it was concluded that these studies do not support the concept of a microclimate of distinctive pH at the epithelial surface as a determinant of weak electrolyte transport. | Intestinal transport of weak electrolytes: Determinants of influx at the luminal surface. The determinants of weak electrolyte influx into everted segments of rat small intestine have been studied. Preliminary experiments showed that the observed influxes could be described as unidirectional, diffusional fluxes of the nonionized compound uncomplicated by a parallel ionic component. It is shown that the determinants of weak electrolyte influx in this situation may be described in terms of the resistance of the unstirred layer to movement from the bulk phase to the cell surface, the degree of ionization of the weak electrolyte at the cell surface, and the cellular permeability to the nonionized weak electrolyte. Quantitative considerations indicated that the unstirred layer was totally rate-limiting in the cases of some poorly ionized, or highly permeant compounds, but the unstirred layer was not totally rate limiting for most of the compounds studied. Calculation of cellular permeabilities for the nonionized forms of weak electrolytes required assumptions to be made concerning the pH value in the surface fluid layer. A uniform set of permeability data including both weak acids and weak bases was obtained only when it was assumed that the pH in the surface fluid layer was equal to that in the bulk phase, and it was concluded that these studies do not support the concept of a microclimate of distinctive pH at the epithelial surface as a determinant of weak electrolyte transport. | [
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PMID:25951 | Double mode of action of black widow spider venom on frog neuromuscular junction. | Black widow spider venom (BWSV) contains a toxin, alpha-latrotoxin, which is capable of stimulating vesicle release, resulting eventually in depletion of vesicles and block of neuromuscular transmission at the frog neuromuscular junction. Since it has been shown that alpha-latrotoxin very markedly increases the cation conductance of artificial lipid bilayers, it was postulated that BWSV stimulates release by opening channels permeable to Ca2+ and, in the case of Ca2+-free Ringer's, to Na+ which would release Ca2+ from intracellular stores. To test this hypothesis we chose as a sodium substitute, glucosamine, which is impermeable to the venom-induced channels in the lipid bilayers and to the postsynaptic membrane of the frog neuromuscular junction. Electron microscopical analysis showed that up to 75 min perfusion in Na+ and Ca2+-free medium did not alter the ultrastructure of the nerve terminals. However when BWSV was applied in this medium a significant depletion was noticeable within 15 min and after 60 min the terminals were depleted of vesicles whereas the mitochondria were unchanged in number and structure. If BWSV is applied for 60 min in glucosamine Ringer's containing 1.8 mM Ca2+, most of the nerve terminals still have synaptic vesicles scattered in the cytoplasm or clustered around amorphous structures and the mitochondria are swollen. Application of large doses of BWSV in low Ca2+ Ringer's leads to damage of the mitochondria and to very pronounced swelling of the nerve endings, whereas this is not observed if the dose of venom is applied in Na+-free and Ca2+-free Ringer's. Electrophysiological recording showed that neuromuscular transmission is already blocked after 15 min treatment with BWSV in glucosamine-Ringer's. From these results we conclude that BWSV increases the conductance of the nerve terminal membrane to cations such as Na+ and Ca2+ and stimulates release by a mechanism which may not involve its ionophore property. | Double mode of action of black widow spider venom on frog neuromuscular junction. Black widow spider venom (BWSV) contains a toxin, alpha-latrotoxin, which is capable of stimulating vesicle release, resulting eventually in depletion of vesicles and block of neuromuscular transmission at the frog neuromuscular junction. Since it has been shown that alpha-latrotoxin very markedly increases the cation conductance of artificial lipid bilayers, it was postulated that BWSV stimulates release by opening channels permeable to Ca2+ and, in the case of Ca2+-free Ringer's, to Na+ which would release Ca2+ from intracellular stores. To test this hypothesis we chose as a sodium substitute, glucosamine, which is impermeable to the venom-induced channels in the lipid bilayers and to the postsynaptic membrane of the frog neuromuscular junction. Electron microscopical analysis showed that up to 75 min perfusion in Na+ and Ca2+-free medium did not alter the ultrastructure of the nerve terminals. However when BWSV was applied in this medium a significant depletion was noticeable within 15 min and after 60 min the terminals were depleted of vesicles whereas the mitochondria were unchanged in number and structure. If BWSV is applied for 60 min in glucosamine Ringer's containing 1.8 mM Ca2+, most of the nerve terminals still have synaptic vesicles scattered in the cytoplasm or clustered around amorphous structures and the mitochondria are swollen. Application of large doses of BWSV in low Ca2+ Ringer's leads to damage of the mitochondria and to very pronounced swelling of the nerve endings, whereas this is not observed if the dose of venom is applied in Na+-free and Ca2+-free Ringer's. Electrophysiological recording showed that neuromuscular transmission is already blocked after 15 min treatment with BWSV in glucosamine-Ringer's. From these results we conclude that BWSV increases the conductance of the nerve terminal membrane to cations such as Na+ and Ca2+ and stimulates release by a mechanism which may not involve its ionophore property. | [
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PMID:25954 | N-Halo derivatives V: Comparative antimicrobial activity of soft N-chloramine systems. | Comparative antimicrobial activity studies for certain new classes of soft N-chloramines derived from alpha-aminiisobutyric acid and 2-amino-2-methyl-1-propanol were examined using the minimum inhibitory concentration (MIC) and/or the contact germicidal efficiency (CGE) procedures. Several factors significantly aliphatic chain length in a homologous series, (b) the degree of chlorination of thenitrogen atom, (c) the solution pH, (d) the presence of a denaturant, and (e) the nature of a positive charge. | N-Halo derivatives V: Comparative antimicrobial activity of soft N-chloramine systems. Comparative antimicrobial activity studies for certain new classes of soft N-chloramines derived from alpha-aminiisobutyric acid and 2-amino-2-methyl-1-propanol were examined using the minimum inhibitory concentration (MIC) and/or the contact germicidal efficiency (CGE) procedures. Several factors significantly aliphatic chain length in a homologous series, (b) the degree of chlorination of thenitrogen atom, (c) the solution pH, (d) the presence of a denaturant, and (e) the nature of a positive charge. | [
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PMID:25955 | Stability of aqueous solutions of mibolerone. | The kinetics and mechanism of degradation of mibolerone were studied in aqueous buffered solutions in the pH range of 1-8 at 67.5 degrees. Mibolerone showed maximum stability between pH 5.5 and 6.4. At pH 1-2, the major degradative pathway was dehydration followed by migration of the 18-methyl group to form 7alpha,17,17-trimethylgona-4,13-dien-3-one. While there was only one degradation product at pH 1-2, the degradation at pH 7-8 was complex. As many as 12 degradation products were detected by GLC. Mass spectral data indicated that the majority of these products were either oxidation products or isomers. At pH 7.6, the apparent first-order rate constants exhibited marked dependency on buffer concentration. Incorporation of a sequestering agent into the solutions eliminated this dependency, suggesting that trace metal impurities from the buffer reagents were catalyzing the degradation. This was confirmed by degradation studies of solutions in water for injection containing 5 ppm of trace metal ions. Sn+2, Cu"2, and Fe+2 accelerated the degradation, with Fe+2 having the most catalytic effect. The temperature dependence of the rate of degradation was studied in 0.05 M phosphate buffer at pH 6.4. The activation energy was 19.6 +/- 1.63 kcal/mole. | Stability of aqueous solutions of mibolerone. The kinetics and mechanism of degradation of mibolerone were studied in aqueous buffered solutions in the pH range of 1-8 at 67.5 degrees. Mibolerone showed maximum stability between pH 5.5 and 6.4. At pH 1-2, the major degradative pathway was dehydration followed by migration of the 18-methyl group to form 7alpha,17,17-trimethylgona-4,13-dien-3-one. While there was only one degradation product at pH 1-2, the degradation at pH 7-8 was complex. As many as 12 degradation products were detected by GLC. Mass spectral data indicated that the majority of these products were either oxidation products or isomers. At pH 7.6, the apparent first-order rate constants exhibited marked dependency on buffer concentration. Incorporation of a sequestering agent into the solutions eliminated this dependency, suggesting that trace metal impurities from the buffer reagents were catalyzing the degradation. This was confirmed by degradation studies of solutions in water for injection containing 5 ppm of trace metal ions. Sn+2, Cu"2, and Fe+2 accelerated the degradation, with Fe+2 having the most catalytic effect. The temperature dependence of the rate of degradation was studied in 0.05 M phosphate buffer at pH 6.4. The activation energy was 19.6 +/- 1.63 kcal/mole. | [
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PMID:25957 | Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiace and skeletal muscles. | 1. The effects of decreasing pH from 7.40 to 6.20 on the tension developed by direct activation of the myofilaments and by Ca2+ release from the sarcoplasmic reticulum were studied comparatively in segments of single cells of skeletal muscle (frog semitendinosus) and cardiac muscle (rat ventricle) from which the sarcolemma had been removed by micro-dissection (skinned muscle cells). 2. The concentration of free Ca2+ in the solutions was buffered with ethylene glycol-bis (beta-aminoethylether N,N'-tetraacetic acid (EGTA). The change of the buffer capacity of a given [total EGTA] caused by varying pH and the uncertainty about the value of the equilibrium constant for Ca-EGTA have been taken into account in the interpretation of the results. 3. Decreasing pH from 7.40 to 6.20 produced an increase in the [free Ca2+] required for the myofilaments to develop 50% of the maximum tension by a factor of about 5 in skinned cardiac cells but of only 3 in skeletal muscle fibres. In addition, acidosis depressed the maximum tension developed in the presence of a saturating [free Ca2+] by approximately the same amount in the two tissues. 4. The pH optimum for loading the sarcoplasmic reticulum of skinned fibres from skeletal muscle decreased when the pCa (-log [free Ca2+]) in the loading solution decreased. The optimum was pH 7.40-7.00 for a loading at pCa 7.75, pH 7.00-6.60 at pCa 7.00 and pH 6.60-6.20 at pCa 6.00. 5. The pH optimum for loading the sarcoplasmic reticulum of skinned cardiac cells with a solution at pCa 7.75 was about pH 7.40 as in skeletal muscle fibres. But the cardiac sarcoplasmic reticulum could not be loaded with a [free Ca2+] much higher than pCa 7.75 because a higher [free Ca2+] triggered a Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum. 6. The pH optimum of about 7.40 for the loading of the cardiac sarcoplasmic reticulum was also optimum for the Ca2+-induced release of Ca2+ from it. 7. It was concluded that the effects of acidosis on the cardiac sarcoplasmic reticulum accentuate the depressive action of decreasing pH on the myofilaments. This may explain the pronounced depression of contractility observed during acidosis in cardiac muscle. In contrast, a moderate acidosis causes an effect on skeletal muscle sarcoplasmic reticulum that could compensate for the depressive action on the myofilaments, which is, in addition, less pronounced than in cardiac muscle. | Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiace and skeletal muscles. 1. The effects of decreasing pH from 7.40 to 6.20 on the tension developed by direct activation of the myofilaments and by Ca2+ release from the sarcoplasmic reticulum were studied comparatively in segments of single cells of skeletal muscle (frog semitendinosus) and cardiac muscle (rat ventricle) from which the sarcolemma had been removed by micro-dissection (skinned muscle cells). 2. The concentration of free Ca2+ in the solutions was buffered with ethylene glycol-bis (beta-aminoethylether N,N'-tetraacetic acid (EGTA). The change of the buffer capacity of a given [total EGTA] caused by varying pH and the uncertainty about the value of the equilibrium constant for Ca-EGTA have been taken into account in the interpretation of the results. 3. Decreasing pH from 7.40 to 6.20 produced an increase in the [free Ca2+] required for the myofilaments to develop 50% of the maximum tension by a factor of about 5 in skinned cardiac cells but of only 3 in skeletal muscle fibres. In addition, acidosis depressed the maximum tension developed in the presence of a saturating [free Ca2+] by approximately the same amount in the two tissues. 4. The pH optimum for loading the sarcoplasmic reticulum of skinned fibres from skeletal muscle decreased when the pCa (-log [free Ca2+]) in the loading solution decreased. The optimum was pH 7.40-7.00 for a loading at pCa 7.75, pH 7.00-6.60 at pCa 7.00 and pH 6.60-6.20 at pCa 6.00. 5. The pH optimum for loading the sarcoplasmic reticulum of skinned cardiac cells with a solution at pCa 7.75 was about pH 7.40 as in skeletal muscle fibres. But the cardiac sarcoplasmic reticulum could not be loaded with a [free Ca2+] much higher than pCa 7.75 because a higher [free Ca2+] triggered a Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum. 6. The pH optimum of about 7.40 for the loading of the cardiac sarcoplasmic reticulum was also optimum for the Ca2+-induced release of Ca2+ from it. 7. It was concluded that the effects of acidosis on the cardiac sarcoplasmic reticulum accentuate the depressive action of decreasing pH on the myofilaments. This may explain the pronounced depression of contractility observed during acidosis in cardiac muscle. In contrast, a moderate acidosis causes an effect on skeletal muscle sarcoplasmic reticulum that could compensate for the depressive action on the myofilaments, which is, in addition, less pronounced than in cardiac muscle. | [
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PMID:25959 | The effects of pH and curare on the time course of end-plate currents at the neuromuscular junction of the frog. | 1. The effect of pH changes on synaptic currents has been analysed by external recording of the miniature end-plate currents (m.e.p.c.s) or by recording in voltage-clamped end-plates the current elicited by nerve stimulation (e.p.c.). 2. Changes in pH do not appreciably effect the peak amplitude of the current produced by a single quantum or by short ionophoretic pulses of acetylcholine. 3. The time constant of decay of the m.e.p.c.s is prolonged by about 50% in acid pH and shortened by about the same amount in alkaline pH. This effect is independent of the cholinesterase activity of the end-plate. 4. In curarized preparations the decay of the e.p.c. is shorter than in Mg-blocked end-plate even in the absence of cholinesterase blocking agents. 5. The action of pH on the decays can be explained by a titration of the surface charges of the membrane which effects the voltage dependent reaction that controls the rate of closing of the synaptic channels. | The effects of pH and curare on the time course of end-plate currents at the neuromuscular junction of the frog. 1. The effect of pH changes on synaptic currents has been analysed by external recording of the miniature end-plate currents (m.e.p.c.s) or by recording in voltage-clamped end-plates the current elicited by nerve stimulation (e.p.c.). 2. Changes in pH do not appreciably effect the peak amplitude of the current produced by a single quantum or by short ionophoretic pulses of acetylcholine. 3. The time constant of decay of the m.e.p.c.s is prolonged by about 50% in acid pH and shortened by about the same amount in alkaline pH. This effect is independent of the cholinesterase activity of the end-plate. 4. In curarized preparations the decay of the e.p.c. is shorter than in Mg-blocked end-plate even in the absence of cholinesterase blocking agents. 5. The action of pH on the decays can be explained by a titration of the surface charges of the membrane which effects the voltage dependent reaction that controls the rate of closing of the synaptic channels. | [
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PMID:25960 | Effects of dithiothreitol on end-plate currents. | 1. End-plate currents have been studied in frog cutaneus pectoris nerve-muscle preparations mounted in continuously flowing solution, using the voltage clamp technique. 2. Exposure of the muscle to 1 mM-dithiothreitol reduced the amplitude of end-plate currents by a factor of 2.7 (mean; range 1.6-3.4; twelve fibres). 3. 1 mM-dithiothreitol also caused a 2.7-fold (2.3-3.1) increase in the rate of decay, and a 1.4-fold (1.3-1.6) decrease in the time to peak of end-plate currents. During the onset of action of dithiothreitol, there was little or no indication of departure of end-plate current decay from a simple exponential. 4. Dithiothreitol actions on amplitude and decay of end-plate currents developed with similar time courses and both effects were slower in onset at pH 7.2 than at pH 8.5. 5. The actions of dithiothreitol were reversed by exposure of the muscle to 1 mM-5,5'-dithio-bis-(2-nitrobenzoic acid). 6. Following dithiothreitol treatment, the rates of decay of end-plate currents continued to depend on membrane potential; there was little or no change in the slope of the relation between in (rate of decay) and membrane potential, consistent with little or no change in the dipole moment of a gating molecule for ion channels. 7. Dithiothreitol changed the relation between peak end-plate current and membrane potential, so that peak conductance increased at more negative membrane potentials; this finding could be accounted for in terms of the closure of ion-channel gates becoming faster though remaining voltage-sensitive after exposure to dithiothreitol. 8. It is concluded that dithiothreitol causes changes in the kinetics of gating of ion channels associated with receptors and that these changes accompany changes in the binding of ACh to receptors. | Effects of dithiothreitol on end-plate currents. 1. End-plate currents have been studied in frog cutaneus pectoris nerve-muscle preparations mounted in continuously flowing solution, using the voltage clamp technique. 2. Exposure of the muscle to 1 mM-dithiothreitol reduced the amplitude of end-plate currents by a factor of 2.7 (mean; range 1.6-3.4; twelve fibres). 3. 1 mM-dithiothreitol also caused a 2.7-fold (2.3-3.1) increase in the rate of decay, and a 1.4-fold (1.3-1.6) decrease in the time to peak of end-plate currents. During the onset of action of dithiothreitol, there was little or no indication of departure of end-plate current decay from a simple exponential. 4. Dithiothreitol actions on amplitude and decay of end-plate currents developed with similar time courses and both effects were slower in onset at pH 7.2 than at pH 8.5. 5. The actions of dithiothreitol were reversed by exposure of the muscle to 1 mM-5,5'-dithio-bis-(2-nitrobenzoic acid). 6. Following dithiothreitol treatment, the rates of decay of end-plate currents continued to depend on membrane potential; there was little or no change in the slope of the relation between in (rate of decay) and membrane potential, consistent with little or no change in the dipole moment of a gating molecule for ion channels. 7. Dithiothreitol changed the relation between peak end-plate current and membrane potential, so that peak conductance increased at more negative membrane potentials; this finding could be accounted for in terms of the closure of ion-channel gates becoming faster though remaining voltage-sensitive after exposure to dithiothreitol. 8. It is concluded that dithiothreitol causes changes in the kinetics of gating of ion channels associated with receptors and that these changes accompany changes in the binding of ACh to receptors. | [
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PMID:25961 | Anion transport of the red cell under non-equilibrium conditions. | 1. The exchange of sulphate for chloride across the human red cell membrane was measured in both directions, i.e. by sulphate influx and sulphate efflux. The influence of the concomitant transient pH changes was minimized by phosphate buffer and by choosing experimental conditions of moderate pH sensitivity (pH 6.4 and 7.8). Sulphate self exchange was determined in chloride-free erythrocyte suspensions. 2. The transport of external sulphate into red cells proceeded at a 15-fold greater rate if its initial concentration was raised from 5 to 95 mM. In contrast the velocity constant of sulphate efflux into sodium chloride medium increased only twofold when the intracellular sulphate concentration was increased. To explain this asymmetry it is proposed that external sulphate ions are more able to complete with chloride for the anion transport sites that those present in the cell interior. 3. The transient membrane potential due to the uneven distribution of sulphate and chloride was shown by the rapid introduction of chromate into the cells. When the erythrocytes contained chloride and the external anion was sulphate, the cells took up chromate 50 times (16.5 degrees C) faster than with equilibrium chloride distribution. 4. Chloride efflux into sodium sulphate media was measured by a chloride-sensitive electrode. Under buffered conditions in neutral and alkaline media two kinetic components were observed as the result of chloride exchange against hydroxyl and sulphate ions. At pH 6.4, chloride efflux was characterized by a single velocity constant identical to that of sulphate movement in the opposite direction. The results show that under appropriate circumstances net chloride efflux measurements can provide comparative data on the anion permeability of the red cell membrane. | Anion transport of the red cell under non-equilibrium conditions. 1. The exchange of sulphate for chloride across the human red cell membrane was measured in both directions, i.e. by sulphate influx and sulphate efflux. The influence of the concomitant transient pH changes was minimized by phosphate buffer and by choosing experimental conditions of moderate pH sensitivity (pH 6.4 and 7.8). Sulphate self exchange was determined in chloride-free erythrocyte suspensions. 2. The transport of external sulphate into red cells proceeded at a 15-fold greater rate if its initial concentration was raised from 5 to 95 mM. In contrast the velocity constant of sulphate efflux into sodium chloride medium increased only twofold when the intracellular sulphate concentration was increased. To explain this asymmetry it is proposed that external sulphate ions are more able to complete with chloride for the anion transport sites that those present in the cell interior. 3. The transient membrane potential due to the uneven distribution of sulphate and chloride was shown by the rapid introduction of chromate into the cells. When the erythrocytes contained chloride and the external anion was sulphate, the cells took up chromate 50 times (16.5 degrees C) faster than with equilibrium chloride distribution. 4. Chloride efflux into sodium sulphate media was measured by a chloride-sensitive electrode. Under buffered conditions in neutral and alkaline media two kinetic components were observed as the result of chloride exchange against hydroxyl and sulphate ions. At pH 6.4, chloride efflux was characterized by a single velocity constant identical to that of sulphate movement in the opposite direction. The results show that under appropriate circumstances net chloride efflux measurements can provide comparative data on the anion permeability of the red cell membrane. | [
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PMID:25967 | 5-Chloro-2-phenyl-1-benzo[b]thiophene-3-alkanimines, potential antipsychotic agents. | The title compounds (most notably 2a) were synthesized on the basis of the N-methylation hypothesis of schizophrenia. They were evaluated in dopamine and haloperidol receptor assays. The binding characteristics were comparable in some cases to known neuroleptics. | 5-Chloro-2-phenyl-1-benzo[b]thiophene-3-alkanimines, potential antipsychotic agents. The title compounds (most notably 2a) were synthesized on the basis of the N-methylation hypothesis of schizophrenia. They were evaluated in dopamine and haloperidol receptor assays. The binding characteristics were comparable in some cases to known neuroleptics. | [
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PMID:25968 | Chemical and electrochemical oxidation of 7-hydroxychlorpromazine. | The oxidation of 7-hydroxychlorpromazine, a process associated with several side effects of chlorpromazine therapy, was examined in vitro by electrochemistry and rapid-scanning spectrophotometry. At pH 2, the oxidation results in a quantitative yield of 7,8-dioxochlorpromazine, but several intermediates are observable during the course of the reaction. These include a quinone imine with a half-life of 0.1 s, a monosubstituted benzoquinone with a half-life of approximately 50 s, and a disubstituted benzoquinone with a half-life of approximately 5 min. The concentrations of each intermediate were determined quantitatively as a function of time, and a complete oxidation mechanism is proposed. At pH 7, the yield of 7,8-dioxochlorpromazine is less than at pH 2, and an additional reaction pathway involving direct hydroxylation of the quinone imine is observed. The relationship of these reactions to the pharmacology of the hydroxylated chlorpromazine metabolites is discussed. | Chemical and electrochemical oxidation of 7-hydroxychlorpromazine. The oxidation of 7-hydroxychlorpromazine, a process associated with several side effects of chlorpromazine therapy, was examined in vitro by electrochemistry and rapid-scanning spectrophotometry. At pH 2, the oxidation results in a quantitative yield of 7,8-dioxochlorpromazine, but several intermediates are observable during the course of the reaction. These include a quinone imine with a half-life of 0.1 s, a monosubstituted benzoquinone with a half-life of approximately 50 s, and a disubstituted benzoquinone with a half-life of approximately 5 min. The concentrations of each intermediate were determined quantitatively as a function of time, and a complete oxidation mechanism is proposed. At pH 7, the yield of 7,8-dioxochlorpromazine is less than at pH 2, and an additional reaction pathway involving direct hydroxylation of the quinone imine is observed. The relationship of these reactions to the pharmacology of the hydroxylated chlorpromazine metabolites is discussed. | [
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PMID:25970 | The incidence of mosquitoes feeding on mothers and babies at Kisumu, Kenya. | Blood fed mosquitoes were collected inside four bed nets in which mother/child pairs, having different haptoglobin types, slept. The bloodmeals were analysed by gradient gel electrophoresis to determine on which person the mosquitoes had fed. The results suggest that the mothers are fed on much more than babies by both Anopheles gambiae s.l. and Culex fatigans. In one hut at least 15.2 per cent of the mosquitoes had taken all or part of their bloodmeal elsewhere. A greater number of double feeds were detected from engorged Cx. fatigans (12.6%) than An. gambiae s.l. (2.7%). | The incidence of mosquitoes feeding on mothers and babies at Kisumu, Kenya. Blood fed mosquitoes were collected inside four bed nets in which mother/child pairs, having different haptoglobin types, slept. The bloodmeals were analysed by gradient gel electrophoresis to determine on which person the mosquitoes had fed. The results suggest that the mothers are fed on much more than babies by both Anopheles gambiae s.l. and Culex fatigans. In one hut at least 15.2 per cent of the mosquitoes had taken all or part of their bloodmeal elsewhere. A greater number of double feeds were detected from engorged Cx. fatigans (12.6%) than An. gambiae s.l. (2.7%). | [
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PMID:25971 | Experience with 425 subfertile male patients. | Subfertility was evaluated in 425 men. Varicocele were diagnosed in 37.4 per cent of these patients and surgical correction of the varicocele in 68 men resulted in a 65 per cent improvement in semen quality. Idiopathic abnormalities were found in 25.4 per cent of the patients. Since the study group was heterogenous setereotyped or empirical treatment cannot hope to be successful if uniformly applied. Other specific causes of male subfertility are identified and therapeutic options are discussed. | Experience with 425 subfertile male patients. Subfertility was evaluated in 425 men. Varicocele were diagnosed in 37.4 per cent of these patients and surgical correction of the varicocele in 68 men resulted in a 65 per cent improvement in semen quality. Idiopathic abnormalities were found in 25.4 per cent of the patients. Since the study group was heterogenous setereotyped or empirical treatment cannot hope to be successful if uniformly applied. Other specific causes of male subfertility are identified and therapeutic options are discussed. | [
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PMID:25973 | Effects of Kö 1366 on the hemodynamics of the normotensive and hypertensive elderly subjects. | The hemodynamic effects of a new adrenergic beta-receptor blocking agent, Kö 1366 were investigated in normotensive and hypertensive elderly subjects. This study, in its important part, was carried out to clarify whether the intrinsic sympathomimetic activity of Kö 1366 modified the hemodynamic changes due to beta-adrenergic blocking action or not. Hemodynamic changes elicited by intravenously administered Kö 1366 (0.05 mg/Kg) were correlated with age, cardiac index, and electrocardiographic findings, respectively. With reference to age in normotensive subjects, Kö 1366 caused no significant differences in rate of changes of various hemodynamic items between 3 age groups. Normotensive elderly subjects were divided into 3 groups according to cardic index. Cardiac index decreased greatly in high cardiac index group, slightly in normal cardiac index group, whereas it increased greatly in low cardiac index group. Stroke volume index showed the same tendency as cardiac index. Hypertensive elderly subjects were, also divided into 2 groups according to cardiac index. Again, cardiac index decreased in higher cardiac index group, while it increased in lower cardiac index group, though a statistical evaluation showed only a tendency of significant difference between 2 groups. In the next, hypertensive elderly subjects were divided into 2 groups according to electrocardiographic findings of left ventricular hypertrophy. Cardiac index decreased in the subjects without electrocardiographic changes, while it increased in the subjects with electrocardiographic changes. Stroke volume index of both groups increased: a greater increase in the subjects with electrocardiographic changes and a tendency of significant difference between 2 groups was recognized. These results suggested that Kö 1366 exhibited the intrinsic symathomimetic activity on the inotropism in the hearts with depressed contractile function of both normotensive and hypertensive subjects, thereby canceling the negative chronotropic action of this agent. This could be a more advantageous point of Kö 1366 in the treatment of elderly subjects with cardiac diseases than other agents without the intrinsic sympathomimetic activity. | Effects of Kö 1366 on the hemodynamics of the normotensive and hypertensive elderly subjects. The hemodynamic effects of a new adrenergic beta-receptor blocking agent, Kö 1366 were investigated in normotensive and hypertensive elderly subjects. This study, in its important part, was carried out to clarify whether the intrinsic sympathomimetic activity of Kö 1366 modified the hemodynamic changes due to beta-adrenergic blocking action or not. Hemodynamic changes elicited by intravenously administered Kö 1366 (0.05 mg/Kg) were correlated with age, cardiac index, and electrocardiographic findings, respectively. With reference to age in normotensive subjects, Kö 1366 caused no significant differences in rate of changes of various hemodynamic items between 3 age groups. Normotensive elderly subjects were divided into 3 groups according to cardic index. Cardiac index decreased greatly in high cardiac index group, slightly in normal cardiac index group, whereas it increased greatly in low cardiac index group. Stroke volume index showed the same tendency as cardiac index. Hypertensive elderly subjects were, also divided into 2 groups according to cardiac index. Again, cardiac index decreased in higher cardiac index group, while it increased in lower cardiac index group, though a statistical evaluation showed only a tendency of significant difference between 2 groups. In the next, hypertensive elderly subjects were divided into 2 groups according to electrocardiographic findings of left ventricular hypertrophy. Cardiac index decreased in the subjects without electrocardiographic changes, while it increased in the subjects with electrocardiographic changes. Stroke volume index of both groups increased: a greater increase in the subjects with electrocardiographic changes and a tendency of significant difference between 2 groups was recognized. These results suggested that Kö 1366 exhibited the intrinsic symathomimetic activity on the inotropism in the hearts with depressed contractile function of both normotensive and hypertensive subjects, thereby canceling the negative chronotropic action of this agent. This could be a more advantageous point of Kö 1366 in the treatment of elderly subjects with cardiac diseases than other agents without the intrinsic sympathomimetic activity. | [
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PMID:25987 | [Metabolic changes in the retina after experimental microembolism in the miniature pig (author's transl)]. | Experimental microembolisation provokes hemodynamics and metabolic changes in the inner retina. The metabolic modifications are the consequence of focal tissue ischaemia and have as a main feature, the activation of anaerobic glycolysis. This activation produces an increase in the production of lactic acid which, in turn, causes a drop of tissular pH. Choroidal circulation maintains photoreceptors in a physiological state. | [Metabolic changes in the retina after experimental microembolism in the miniature pig (author's transl)]. Experimental microembolisation provokes hemodynamics and metabolic changes in the inner retina. The metabolic modifications are the consequence of focal tissue ischaemia and have as a main feature, the activation of anaerobic glycolysis. This activation produces an increase in the production of lactic acid which, in turn, causes a drop of tissular pH. Choroidal circulation maintains photoreceptors in a physiological state. | [
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PMID:26006 | [Apparatus for the multichannel pH measurement of the upper section of the gastrointestinal tract]. | To study simultaneously the acid-base processes in the esophagus, stomach and duodenum a procedure involving the use of multichannel pH measurements is proposed and a setup designed whose basic elements are a 6-channel pH-probe and a registering device. A visual and graphic registration of the investigation results is envisaged. | [Apparatus for the multichannel pH measurement of the upper section of the gastrointestinal tract]. To study simultaneously the acid-base processes in the esophagus, stomach and duodenum a procedure involving the use of multichannel pH measurements is proposed and a setup designed whose basic elements are a 6-channel pH-probe and a registering device. A visual and graphic registration of the investigation results is envisaged. | [
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PMID:26014 | Tardive dyskinesia and antihistamines. | A case of tardive dyskinesia due to the prolonged administration of antihistamines is reported. Clinical features are discussed, as well as the alleviation of these by haloperidol. | Tardive dyskinesia and antihistamines. A case of tardive dyskinesia due to the prolonged administration of antihistamines is reported. Clinical features are discussed, as well as the alleviation of these by haloperidol. | [
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PMID:26015 | Labetalol in the treatment of hypertensive renal patients. | The efficacy of labetalol in lowering blood pressure was assessed in 18 patients with chronic renal failure and hypertension. Before the start of labetalol therapy, all patients were receiving combined antihypertensive therapy, the most common being a beta-blocker and hydrallazine. Over the period of about four weeks labetalol was substituted for the prior therapy. 51Cr edetic acid (EDTA) estimations of glomerular filtration rate were performed before labetalol therapy, and then again after one and six months. Before the therapy with labetalol, 12 of the 18 patients had supine diastolic blood pressures of 100 mm Hg or more. At six months, 14 patients remained in the trial and, of these, only four had a supine diastolic blood pressure of 100 mm Hg or more. In the supine position there was a significant reduction of systolic, but not of diastolic, blood pressure. However, in the erect position there was a significant reduction both in systolic and in diastolic blood presure. Pulse rate did not vary significantly. Few side effects were encountered, transient postural dizziness being the most common side effect. Labetalol seems to be an effective substitute for the beta-blocker plus hydrallazine therapy. However, it is not as potent as minoxidil. | Labetalol in the treatment of hypertensive renal patients. The efficacy of labetalol in lowering blood pressure was assessed in 18 patients with chronic renal failure and hypertension. Before the start of labetalol therapy, all patients were receiving combined antihypertensive therapy, the most common being a beta-blocker and hydrallazine. Over the period of about four weeks labetalol was substituted for the prior therapy. 51Cr edetic acid (EDTA) estimations of glomerular filtration rate were performed before labetalol therapy, and then again after one and six months. Before the therapy with labetalol, 12 of the 18 patients had supine diastolic blood pressures of 100 mm Hg or more. At six months, 14 patients remained in the trial and, of these, only four had a supine diastolic blood pressure of 100 mm Hg or more. In the supine position there was a significant reduction of systolic, but not of diastolic, blood pressure. However, in the erect position there was a significant reduction both in systolic and in diastolic blood presure. Pulse rate did not vary significantly. Few side effects were encountered, transient postural dizziness being the most common side effect. Labetalol seems to be an effective substitute for the beta-blocker plus hydrallazine therapy. However, it is not as potent as minoxidil. | [
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PMID:26012 | [Acidophilic thermotolerant Candida utilis yeast strain obtained by continuous cultivation]. | An acidophilic thermotolerant strain of Candida utilis 1668-3-37 was produced under conditions of continuous cultivation during 128 days. The optimum pH is 3.0-4.5 at 32 degrees C. The strain grows with mumax=0.30 hr(-1) at 37-38 degrees C and the same pH values if dry yeast autolysate (0.05%) is added to the medium. | [Acidophilic thermotolerant Candida utilis yeast strain obtained by continuous cultivation]. An acidophilic thermotolerant strain of Candida utilis 1668-3-37 was produced under conditions of continuous cultivation during 128 days. The optimum pH is 3.0-4.5 at 32 degrees C. The strain grows with mumax=0.30 hr(-1) at 37-38 degrees C and the same pH values if dry yeast autolysate (0.05%) is added to the medium. | [
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PMID:26013 | [Metal content in Bacillus megaterium cells under different cultivation conditions]. | The content of K, Na, Mg, Fe, Al, Ba, Sr and Cu was studied in the cells of a chemostal culture of Bacillus megaterium whose growth was limited with citrate, the growth rates being D=mu=0.2, 0.4 and 0.7 hr(-1). The growth was affected also by acid and alkaline values of pH. The content of metals in the cells could change tenfold, depending on their physiological state. The content of Ca, Mg and Fe increased with the growth rate while that of K remained constant. The content of metals changed even more sharply, depending on pH. In the alkaline medium, the content of K, Na, Mg, Ca and Fe decreased, in contrast to that of Al, Ba, Sr and Cu, probably as a result of damaged transport. | [Metal content in Bacillus megaterium cells under different cultivation conditions]. The content of K, Na, Mg, Fe, Al, Ba, Sr and Cu was studied in the cells of a chemostal culture of Bacillus megaterium whose growth was limited with citrate, the growth rates being D=mu=0.2, 0.4 and 0.7 hr(-1). The growth was affected also by acid and alkaline values of pH. The content of metals in the cells could change tenfold, depending on their physiological state. The content of Ca, Mg and Fe increased with the growth rate while that of K remained constant. The content of metals changed even more sharply, depending on pH. In the alkaline medium, the content of K, Na, Mg, Ca and Fe decreased, in contrast to that of Al, Ba, Sr and Cu, probably as a result of damaged transport. | [
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PMID:26019 | Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates. | A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by compentent S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is competitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin. | Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates. A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by compentent S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is competitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin. | [
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PMID:26020 | The properties of mnemiopsin, a bioluminescent and light sensitive protein purified by hollow fiber techniques. | A calcium activated photoprotein, termed mnemiopsin, which emits bioluminescence upon the addition of calcium ion, has been isolated from the Ctenophore, Memiopsis leidyi, and purified by hollow fiber techniques. The system is similar to aequorin, from the jellyfish Aequorea, except that mnemiopsin can be light-inactivated. Separation of mnemiopsin from the dilute and large volume animal homogenate proved difficult with conventional biochemical techniques. A continuous flow process utilizing large surface area hollow fibers for filtration, concentration, and dialysis was developed which may also be applicable to the purification of other proteins. The resulting mnemiopsin concentrate, after further purification, was judged to be about 90% pure by its gel electrophoretic profile. Estimates by molecular sieve chromatography and SDS gel electrophoresis gave a molecular weight of about 23,000 daltons. A calcium specificity for triggering light emission was studied by comparison of triggering with a variety of cations and anions and by investigating the effects of calcium ionophores and antagonists. The activity of mnemiospin was characterized with respect to pH, temperature and ionic strength. The stability of mnemiopsin activity after exposure to proteases, denaturants, protein group specific reagents, detergents, elevated temperatures and light was determined. Some years ago our laboratory reported that the bioluminescence reaction in the ctenophores which had long eluded definition involved a calcium activated photoprotein similar in many respects to that found in other coelenterates, notably Aequorea. We found, moreover, that the systems differed in that the bioluminescent activity of the isolated protein was lost following exposure to light. The purification and characterization of this biochemical system was undertaken both in our laboratory and by Ward and Seliger. These latter reports provide a detailed and firm foundation for the understanding of the components and mechanisms involved. While many of our results are in agreement with theirs, our approaches, inquiries, and results differed in several significant ways, the description of which forms the basis for this report. In particular, we took a different approach in the purification of the Mnemiopsis photoprotein which in itself is rather a formidable task. The technique was successful and may point the way to other applications where large volume dilute solutions prove cumbersome. Secondly, our study of the effects of salts, proteases, detergents, and other agents indicate that the protein, though sensitive to calcium and visible light inactivation, is relatively resistant to some agents which commonly inactivate proteins. | The properties of mnemiopsin, a bioluminescent and light sensitive protein purified by hollow fiber techniques. A calcium activated photoprotein, termed mnemiopsin, which emits bioluminescence upon the addition of calcium ion, has been isolated from the Ctenophore, Memiopsis leidyi, and purified by hollow fiber techniques. The system is similar to aequorin, from the jellyfish Aequorea, except that mnemiopsin can be light-inactivated. Separation of mnemiopsin from the dilute and large volume animal homogenate proved difficult with conventional biochemical techniques. A continuous flow process utilizing large surface area hollow fibers for filtration, concentration, and dialysis was developed which may also be applicable to the purification of other proteins. The resulting mnemiopsin concentrate, after further purification, was judged to be about 90% pure by its gel electrophoretic profile. Estimates by molecular sieve chromatography and SDS gel electrophoresis gave a molecular weight of about 23,000 daltons. A calcium specificity for triggering light emission was studied by comparison of triggering with a variety of cations and anions and by investigating the effects of calcium ionophores and antagonists. The activity of mnemiospin was characterized with respect to pH, temperature and ionic strength. The stability of mnemiopsin activity after exposure to proteases, denaturants, protein group specific reagents, detergents, elevated temperatures and light was determined. Some years ago our laboratory reported that the bioluminescence reaction in the ctenophores which had long eluded definition involved a calcium activated photoprotein similar in many respects to that found in other coelenterates, notably Aequorea. We found, moreover, that the systems differed in that the bioluminescent activity of the isolated protein was lost following exposure to light. The purification and characterization of this biochemical system was undertaken both in our laboratory and by Ward and Seliger. These latter reports provide a detailed and firm foundation for the understanding of the components and mechanisms involved. While many of our results are in agreement with theirs, our approaches, inquiries, and results differed in several significant ways, the description of which forms the basis for this report. In particular, we took a different approach in the purification of the Mnemiopsis photoprotein which in itself is rather a formidable task. The technique was successful and may point the way to other applications where large volume dilute solutions prove cumbersome. Secondly, our study of the effects of salts, proteases, detergents, and other agents indicate that the protein, though sensitive to calcium and visible light inactivation, is relatively resistant to some agents which commonly inactivate proteins. | [
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PMID:26021 | Extraction of tyrosine aminotransferase mRNA by polyribosome immunosorption on sepharose 4B. | Sepharose 4B column with antibody to tyrosine aminotransferase (E.C. 2.6.1.5) (TAT) covalently bound can selectively remove a specific fraction of TAT polysomes from rat liver homogenates. From the polysomes which was adsorbed by immunosorbent one may obtain mRNA containing a segment of poly-adenilate-rich residues, having sedimentation constant of 11--16S. This mRNA may program synthesis of the specific protein in a cell free system. Near 70% of the protein was synthesized in such a system, may react with the antibody to TAT. | Extraction of tyrosine aminotransferase mRNA by polyribosome immunosorption on sepharose 4B. Sepharose 4B column with antibody to tyrosine aminotransferase (E.C. 2.6.1.5) (TAT) covalently bound can selectively remove a specific fraction of TAT polysomes from rat liver homogenates. From the polysomes which was adsorbed by immunosorbent one may obtain mRNA containing a segment of poly-adenilate-rich residues, having sedimentation constant of 11--16S. This mRNA may program synthesis of the specific protein in a cell free system. Near 70% of the protein was synthesized in such a system, may react with the antibody to TAT. | [
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PMID:26028 | The binding of intravenous and oral biliary contrast agents to human and bovine serum albumin. | The binding of two homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3--5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed. | The binding of intravenous and oral biliary contrast agents to human and bovine serum albumin. The binding of two homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3--5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed. | [
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PMID:26036 | [Hemorrhagic rectocolitis. Attempt at programmed therapy]. | Planned treatment of 30 cases of haemorrhagic colitis is reported. Its protocol included salazopyrine and benzodiazepine per os, salazopyrine and prednisone per rectum, and parenteral ACTH depot. Administration for 60 days brought good results in cases of slight or average severity. Recurrences, albeit less severe, were not prevented, however. Attention is drawn to the need to follow up this group of patients for a longer period. Even so, it is felt that the effect of this form of treatment is primarily symptomatic. | [Hemorrhagic rectocolitis. Attempt at programmed therapy]. Planned treatment of 30 cases of haemorrhagic colitis is reported. Its protocol included salazopyrine and benzodiazepine per os, salazopyrine and prednisone per rectum, and parenteral ACTH depot. Administration for 60 days brought good results in cases of slight or average severity. Recurrences, albeit less severe, were not prevented, however. Attention is drawn to the need to follow up this group of patients for a longer period. Even so, it is felt that the effect of this form of treatment is primarily symptomatic. | [
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PMID:26038 | Expression of hormonally induced tyrosine aminotransferase in host liver and Morris hepatoma No. 7777 during cofactor depletion. | Cytoplasmic tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC2.6.1.5) was partially purified from host liver and Morris hepatoma No. 7777 grown in pyridoxine depleted rats. The animals were sacrificed six hours following the intraperitoneal administration of hydrocortisone hemisuccinate. Enzyme preparations were subsequently resolved by electrophoresis on polyacrylamide gels. Enzyme activity was detected histochemically in situ on the gels. Six and at least three enzymatically active protein peaks were detected in host liver and the hepatoma, respectively, by this method. | Expression of hormonally induced tyrosine aminotransferase in host liver and Morris hepatoma No. 7777 during cofactor depletion. Cytoplasmic tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC2.6.1.5) was partially purified from host liver and Morris hepatoma No. 7777 grown in pyridoxine depleted rats. The animals were sacrificed six hours following the intraperitoneal administration of hydrocortisone hemisuccinate. Enzyme preparations were subsequently resolved by electrophoresis on polyacrylamide gels. Enzyme activity was detected histochemically in situ on the gels. Six and at least three enzymatically active protein peaks were detected in host liver and the hepatoma, respectively, by this method. | [
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PMID:26041 | [The detection of bacterial antigens by counter-immunoelectrophoresis in N. meningitidis, H. influenzae serotype b, S. pneumoniae infections. Diagnostic value and evolutive aspect (in 216 cases) (author's transl)]. | Using counterimmunoelectrophoresis (CIE) the authors have assayed for soluble bacterial S. pneumoniae, N meningitidis group A, B, C. H. influenzae type b antigens, biological fluids in 216 patients (meningitis: 136; pneumonia: 76; miscellaneous: 4) during 16 months. Because of heterogeneous recruiting (the bacteriology was carried out by different laboratories) the increase in aetiological diagnosis given by CIE is only statistically valid for the bacteriologic negative group when blind antibiotic therapy had already been given. In this group, CIE makes a notable increase in diagnosis of 22,1 % +/- 10,1 in meningitis and 25,5% +/- 12,7 in pneumonia. Various physiopathological aspects are considered concerning soluble bacterial antigens detection during the course of the disease. This method seems very useful and accurate; and therefore should be used in every microbiologic laboratory. | [The detection of bacterial antigens by counter-immunoelectrophoresis in N. meningitidis, H. influenzae serotype b, S. pneumoniae infections. Diagnostic value and evolutive aspect (in 216 cases) (author's transl)]. Using counterimmunoelectrophoresis (CIE) the authors have assayed for soluble bacterial S. pneumoniae, N meningitidis group A, B, C. H. influenzae type b antigens, biological fluids in 216 patients (meningitis: 136; pneumonia: 76; miscellaneous: 4) during 16 months. Because of heterogeneous recruiting (the bacteriology was carried out by different laboratories) the increase in aetiological diagnosis given by CIE is only statistically valid for the bacteriologic negative group when blind antibiotic therapy had already been given. In this group, CIE makes a notable increase in diagnosis of 22,1 % +/- 10,1 in meningitis and 25,5% +/- 12,7 in pneumonia. Various physiopathological aspects are considered concerning soluble bacterial antigens detection during the course of the disease. This method seems very useful and accurate; and therefore should be used in every microbiologic laboratory. | [
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PMID:26042 | A new method for the purification and identification of covalently closed circular DNA molcules. | A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures. | A new method for the purification and identification of covalently closed circular DNA molcules. A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures. | [
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PMID:26043 | Synthesis and properties of 8-azido-1, N6-etheno adenosine triphosphate--a fluorescent and photosensitive ATP analog. | 8-Azido-1,N6-etheno-ATP--a fluorescent and photoreactive ATP analog has been synthesized and characterized by elementary analysis, thin layer chromatography, infrared spectroscopy, proton resonance spectroscopy, UV absorption spectroscopy, and fluorescence spectroscopy. The photolytical decomposition upon irradiation at different pH values is tested. | Synthesis and properties of 8-azido-1, N6-etheno adenosine triphosphate--a fluorescent and photosensitive ATP analog. 8-Azido-1,N6-etheno-ATP--a fluorescent and photoreactive ATP analog has been synthesized and characterized by elementary analysis, thin layer chromatography, infrared spectroscopy, proton resonance spectroscopy, UV absorption spectroscopy, and fluorescence spectroscopy. The photolytical decomposition upon irradiation at different pH values is tested. | [
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PMID:26044 | Short RNA chains synthesized at low pH are initiated at promoter sites. | Under non optimal conditions- either with limiting substrate concentrations (1) or at low pH (2)- RNA polymerase of Escherichia coli synthesizes very short RNA chains. By sequencing one RNA species synthesized at pH 5.8 upon T7 DNA we were able to demonstrate that under these conditions transcription is initiated at a normal promoter site (here A1) but however is terminated soon afterwards at specific artificial sites not used in vivo. | Short RNA chains synthesized at low pH are initiated at promoter sites. Under non optimal conditions- either with limiting substrate concentrations (1) or at low pH (2)- RNA polymerase of Escherichia coli synthesizes very short RNA chains. By sequencing one RNA species synthesized at pH 5.8 upon T7 DNA we were able to demonstrate that under these conditions transcription is initiated at a normal promoter site (here A1) but however is terminated soon afterwards at specific artificial sites not used in vivo. | [
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] |
PMID:26050 | [Polarographic method of determining phenol in the air]. | A specific, highly sensitive and exact polarographic method of phenol determination in the air is developed. The conditions of sample taking are outlined, and the effect of polarographic solution pH on the height of step and semi-wave potential is verified. The suggested method is readily applicable simple for execution, and provides for a good reproducibility and promptness of determination during productional tests. | [Polarographic method of determining phenol in the air]. A specific, highly sensitive and exact polarographic method of phenol determination in the air is developed. The conditions of sample taking are outlined, and the effect of polarographic solution pH on the height of step and semi-wave potential is verified. The suggested method is readily applicable simple for execution, and provides for a good reproducibility and promptness of determination during productional tests. | [
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PMID:26051 | Minichromosome from BK virus as a template for transcription in vitro. | BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA. | Minichromosome from BK virus as a template for transcription in vitro. BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA. | [
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PMID:26052 | Transglutaminase activity in human lymphocytes: early activation by phytomitogens. | Transglutaminase activity is present in human peripheral lymphocytes and is enhanced up to 15-fold within 10-30 min after treatment of the cells with concanavalin A. Phytohemagglutinin has a similar effect. The enzyme is not detected when intact cells are assayed; it is detected only in cell lysates. Concanavalin A enhances transglutaminase activity only when it is incubated with intact cells; concanavalin A treatment of cell lysates has no effect, alpha-Methyl-D-mannoside specifically inhibits the enhancement of transglutaminase activity in cells treated with concanavalin A results from the specific interaction of the lectin with its saccharide binding site on the cell surface, rather than by direct interaction with the enzyme itself. The increased activity of transglutaminase in cells treated with concanavalin A, as compared to unstimulated cells, is maintained under assay conditions in which saturating levels of Ca2+ are present. Transglutaminase may be involved in early cellular events leading to lymphocyte blastogenesis. | Transglutaminase activity in human lymphocytes: early activation by phytomitogens. Transglutaminase activity is present in human peripheral lymphocytes and is enhanced up to 15-fold within 10-30 min after treatment of the cells with concanavalin A. Phytohemagglutinin has a similar effect. The enzyme is not detected when intact cells are assayed; it is detected only in cell lysates. Concanavalin A enhances transglutaminase activity only when it is incubated with intact cells; concanavalin A treatment of cell lysates has no effect, alpha-Methyl-D-mannoside specifically inhibits the enhancement of transglutaminase activity in cells treated with concanavalin A results from the specific interaction of the lectin with its saccharide binding site on the cell surface, rather than by direct interaction with the enzyme itself. The increased activity of transglutaminase in cells treated with concanavalin A, as compared to unstimulated cells, is maintained under assay conditions in which saturating levels of Ca2+ are present. Transglutaminase may be involved in early cellular events leading to lymphocyte blastogenesis. | [
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PMID:26053 | Topographical analysis of regulatory and metal ion binding sites on glutamine synthetase from Escherichia coli: 13C and 31P nuclear magnetic resonance and fluorescence energy transfer study. | The paramagnetic effect of Mn(II) on (13)C and (31)P nuclear magnetic resonance signals from the [2-(13)C]ATP adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] from Escherichia coli was measured. This effect permitted the determination of distances from the 2-C position and the phosphorus of covalently bound AMP to the two Mn(II) binding sites, n(1) and n(2). Binding of Mn(II) to the n(1) site converts an inactive apo-enzyme to its active form, while the metal ion bound at n(2) occupies the metal-nucleotide substrate site. The distances from Mn(II) at the n(1) and n(2) sites to phosphorus are approximately 10 and approximately 7 A and to the 2-C position of the adenine ring are approximately 12 and approximately 11 A, respectively. The fluorescence energy transfer method was used to determine distances between Co(II) at n(1) and n(2) and the adenylyl site. For this experiment the enzyme was adenylylated with epsilon-ATP. The distances between epsilon-adenine and Co(II) at n(1) and n(2) are approximately 13 and approximately 11 A, respectively. Quantitation of the paramagnetic effect due to Co(II) on the (31)P nuclear magnetic resonance signal yielded values of 8 and 6 A for the distances between the phosphorus of the covalently bound AMP and the n(1) and n(2) sites, respectively. The results reveal that the covalent modification site is very close to the catalytic center of the enzyme. In this study both nuclear magnetic resonance and fluorescence energy transfer techniques have been used to determine distances between the same set of sites on an enzyme surface. | Topographical analysis of regulatory and metal ion binding sites on glutamine synthetase from Escherichia coli: 13C and 31P nuclear magnetic resonance and fluorescence energy transfer study. The paramagnetic effect of Mn(II) on (13)C and (31)P nuclear magnetic resonance signals from the [2-(13)C]ATP adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] from Escherichia coli was measured. This effect permitted the determination of distances from the 2-C position and the phosphorus of covalently bound AMP to the two Mn(II) binding sites, n(1) and n(2). Binding of Mn(II) to the n(1) site converts an inactive apo-enzyme to its active form, while the metal ion bound at n(2) occupies the metal-nucleotide substrate site. The distances from Mn(II) at the n(1) and n(2) sites to phosphorus are approximately 10 and approximately 7 A and to the 2-C position of the adenine ring are approximately 12 and approximately 11 A, respectively. The fluorescence energy transfer method was used to determine distances between Co(II) at n(1) and n(2) and the adenylyl site. For this experiment the enzyme was adenylylated with epsilon-ATP. The distances between epsilon-adenine and Co(II) at n(1) and n(2) are approximately 13 and approximately 11 A, respectively. Quantitation of the paramagnetic effect due to Co(II) on the (31)P nuclear magnetic resonance signal yielded values of 8 and 6 A for the distances between the phosphorus of the covalently bound AMP and the n(1) and n(2) sites, respectively. The results reveal that the covalent modification site is very close to the catalytic center of the enzyme. In this study both nuclear magnetic resonance and fluorescence energy transfer techniques have been used to determine distances between the same set of sites on an enzyme surface. | [
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PMID:26054 | Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis. | Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaCl. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean +/- SEM, 2 +/- 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential. | Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis. Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaCl. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean +/- SEM, 2 +/- 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential. | [
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PMID:26055 | Regulation of glutamine synthetase in cultured 3T3-L1 cells by insulin, hydrocortisone, and dibutyryl cyclic AMP. | The 3T3-L1 mouse fibroblast cell line develops morphological and biochemical characteristics of adipocytes when maintained at confluence. This conversion to adipocytes is accelerated by addition of insulin to the culture medium [Green, H. & Kehinde, O. (1975) Cell 5, 19-27]. During the course of the insulin-mediated adipocyte conversion, the specific activity (units/mg of protein) of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] increases more than 100-fold. The specific activities of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) and glucose-6-P dehydrogenase (D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49) also increase but less dramatically (1.5- to 3-fold). In contrast, confluent cells maintained in the absence of insulin for the same time (12-20 days after confluence) display only minimal increases in the activity of these enzymes. Maintenance of confluent cells in culture medium lacking added L-glutamine has little, if any, effect on glutamine synthetase activity in either control or insulin-treated cultures. Treatment of confluent 3T3-L1 cultures with hydrocortisone (1 mug/ml) for 3 days prior to harvesting results in an increase in glutamine synthetase specific activity of 12-fold for control cultures maintained for 13 days in the absence of insulin and 1.4-fold for adipocyte cultures maintained for 13 days in the presence of insulin (10 mug/ml). Treatment of 3T3-L1 control cells and adipocytes with dibutyryl cyclic AMP (1 mM) plus theophylline (1 mM) decreases the glutamine synthetase specific activity and almost completely reverses the insulin- and hydrocortisone-mediated increases in enzyme activity. In contrast, treatment with dibutyryl cyclic AMP plus theophylline has relatively little effect on the specific activities of hexokinase or glucose-6-P dehydrogenase or on the protein content of the cultures. These data indicate that glutamine synthetase activity is hormonally regulated in 3T3-L1 cells. | Regulation of glutamine synthetase in cultured 3T3-L1 cells by insulin, hydrocortisone, and dibutyryl cyclic AMP. The 3T3-L1 mouse fibroblast cell line develops morphological and biochemical characteristics of adipocytes when maintained at confluence. This conversion to adipocytes is accelerated by addition of insulin to the culture medium [Green, H. & Kehinde, O. (1975) Cell 5, 19-27]. During the course of the insulin-mediated adipocyte conversion, the specific activity (units/mg of protein) of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] increases more than 100-fold. The specific activities of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) and glucose-6-P dehydrogenase (D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49) also increase but less dramatically (1.5- to 3-fold). In contrast, confluent cells maintained in the absence of insulin for the same time (12-20 days after confluence) display only minimal increases in the activity of these enzymes. Maintenance of confluent cells in culture medium lacking added L-glutamine has little, if any, effect on glutamine synthetase activity in either control or insulin-treated cultures. Treatment of confluent 3T3-L1 cultures with hydrocortisone (1 mug/ml) for 3 days prior to harvesting results in an increase in glutamine synthetase specific activity of 12-fold for control cultures maintained for 13 days in the absence of insulin and 1.4-fold for adipocyte cultures maintained for 13 days in the presence of insulin (10 mug/ml). Treatment of 3T3-L1 control cells and adipocytes with dibutyryl cyclic AMP (1 mM) plus theophylline (1 mM) decreases the glutamine synthetase specific activity and almost completely reverses the insulin- and hydrocortisone-mediated increases in enzyme activity. In contrast, treatment with dibutyryl cyclic AMP plus theophylline has relatively little effect on the specific activities of hexokinase or glucose-6-P dehydrogenase or on the protein content of the cultures. These data indicate that glutamine synthetase activity is hormonally regulated in 3T3-L1 cells. | [
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PMID:26062 | Crosslinking of the nearest membrane protein neighbors in ATP depleted, calcium enriched and irreversibly sickled red cells. | ATP depleted or Ca2+ (0.1 mM) enriched normal red cells (greater than 80% echinocytes III) subjected to crosslinking by catalytic oxidation contain a greater than 1,000,000 daltons spectrin rich polymer cleavable by dithiothreitol (DTT) reduction. Similar complex is seen after glutaraldehyde crosslinking suggesting spectrin rearrangement into closer contacts or aggregation. In addition, a non-reducible greater than 1,000,000 daltons polymer is produced in fresh rbc or ghosts by high (greater than 0.5 mM) Ca2+ conc. and ionophore A23187. This complex is attributed to endogenous membrane protein crosslinking, catalyzed by a Ca2+ stimulated rbc transglutamidase. ISCs exhibiting a 4 fold increase in Ca2+ and a decrease in ATP do not exhibit these polymers. However, ISCs have an increased propensity to form the spectrin rich polymer during a subsequent ATP depletion and this is associated with a transformation of greater than 60% ISCs into spheroechinocytes. Similar cells are occasionally noted (greater than 4%) in the densest ISC rich fractions separated from fresh blood. We conclude that neither Ca2+, ATP dependent spectrin aggregation nor a spontaneous irreversible crosslinking underlie the membrane lesion of ISCs. Accelerated calcium gain and ATP depletion in ISCs leads to spectrin rearrangement and transformation of ISCs into spheroechinocytes which may represent an end stage ISC lesion resulting in an ISC removal from circulation. | Crosslinking of the nearest membrane protein neighbors in ATP depleted, calcium enriched and irreversibly sickled red cells. ATP depleted or Ca2+ (0.1 mM) enriched normal red cells (greater than 80% echinocytes III) subjected to crosslinking by catalytic oxidation contain a greater than 1,000,000 daltons spectrin rich polymer cleavable by dithiothreitol (DTT) reduction. Similar complex is seen after glutaraldehyde crosslinking suggesting spectrin rearrangement into closer contacts or aggregation. In addition, a non-reducible greater than 1,000,000 daltons polymer is produced in fresh rbc or ghosts by high (greater than 0.5 mM) Ca2+ conc. and ionophore A23187. This complex is attributed to endogenous membrane protein crosslinking, catalyzed by a Ca2+ stimulated rbc transglutamidase. ISCs exhibiting a 4 fold increase in Ca2+ and a decrease in ATP do not exhibit these polymers. However, ISCs have an increased propensity to form the spectrin rich polymer during a subsequent ATP depletion and this is associated with a transformation of greater than 60% ISCs into spheroechinocytes. Similar cells are occasionally noted (greater than 4%) in the densest ISC rich fractions separated from fresh blood. We conclude that neither Ca2+, ATP dependent spectrin aggregation nor a spontaneous irreversible crosslinking underlie the membrane lesion of ISCs. Accelerated calcium gain and ATP depletion in ISCs leads to spectrin rearrangement and transformation of ISCs into spheroechinocytes which may represent an end stage ISC lesion resulting in an ISC removal from circulation. | [
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