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pubmed23n0001_100

Influence of hematocrit, blood gas tensions, and pH on pressure-flow relations in the isolated canine lung.

An isolated perfused canine lung preparation in which determinants of vascular caliber could be individually controlled was developed. The relation of pulmonary arterial (Pa), venous (PV), and alveolar (PA) pressures was such that Pa greater than PA greater than PV throughout the whole lung. The addition of isoprenaline to the perfusate abolished vascular reactivity. Once stability was reached, vascular cross-sectional area remained acceptably constant for 2.25 hours as judged by normalized conductance. The influence of perfusate hematocrit, blood gas tensions, and pH on pressure-flow relations was then studied in 15 isolated canine lungs. The hematocrit-vascular conductance relation was derived at constant perfusion pressure. Conductance varied linearly with hematocrit over a range of 16.5 to 89.5%. Mean pulmonary arterial blood gas tensions were: PO2 = 121 mm Hg, PCO2 = 28 mm Hg, and pH = 7.46. Acute respiratory acidosis (PO2 = 30 mm Hg, PCO2 = 81 mm Hg, pH = 7.17) and lactic acidosis and hypoxemia (PO2 = 32 mm Hg, PCO2 = 21 mm Hg, pH = 6.96) did not significantly alter this relation. Transformation of the conductance-hematocrit data indicated that hematocrit was the most important determinant of relative apparent viscosity of the blood. Both acute respiratory and lactic acidosis failed to significantly increase relative viscosity within the range of hematocrit usually found in secondary polycythemia.

Influence of hematocrit, blood gas tensions, and pH on pressure-flow relations in the isolated canine lung. An isolated perfused canine lung preparation in which determinants of vascular caliber could be individually controlled was developed. The relation of pulmonary arterial (Pa), venous (PV), and alveolar (PA) pressures was such that Pa greater than PA greater than PV throughout the whole lung. The addition of isoprenaline to the perfusate abolished vascular reactivity. Once stability was reached, vascular cross-sectional area remained acceptably constant for 2.25 hours as judged by normalized conductance. The influence of perfusate hematocrit, blood gas tensions, and pH on pressure-flow relations was then studied in 15 isolated canine lungs. The hematocrit-vascular conductance relation was derived at constant perfusion pressure. Conductance varied linearly with hematocrit over a range of 16.5 to 89.5%. Mean pulmonary arterial blood gas tensions were: PO2 = 121 mm Hg, PCO2 = 28 mm Hg, and pH = 7.46. Acute respiratory acidosis (PO2 = 30 mm Hg, PCO2 = 81 mm Hg, pH = 7.17) and lactic acidosis and hypoxemia (PO2 = 32 mm Hg, PCO2 = 21 mm Hg, pH = 6.96) did not significantly alter this relation. Transformation of the conductance-hematocrit data indicated that hematocrit was the most important determinant of relative apparent viscosity of the blood. Both acute respiratory and lactic acidosis failed to significantly increase relative viscosity within the range of hematocrit usually found in secondary polycythemia.

pubmed23n0001_101

Interaction of the chemoreflex and the pulmonary inflation reflex in the regulation of coronary circulation in conscious dogs.

The interaction of chemoreflex and pulmonary inflation reflex control of the coronary circulation was examined in conscious dogs by comparing the responses to chemoreflex stimulation (intracarotid injection of nicotine) when ventilation was allowed to increase with those when ventilation was controlled. The responses were also compared with those elicited by both forced mechanical and spontaneous hyperinflation. When the heart rate was constant, intracarotidly administered nicotine induced an increase in the depth of respiration followed closely by an increase in late diastolic coronary flow from 48 +/- 2 to 106 +/- 8 ml/min and a reduction in late diastolic coronary resistance from 1.62 +/- 0.08 to 0.78 +/- 0.06 mm Hg/ml min-1. After beta-receptor and cholinergic blockade, a similar coronary dilation in response to nicotine occurred only when ventilation was allowed to increase. However, when ventilation was controlled, intracarotidly administered nicotine increased coronary resistance after combined beta-receptor and cholinergic blockade. The reflex coronary dilation was not observed after carotid sinus nerve section or after alpha-receptor blockade. Thus, nicotine stimulation of the carotid chemoreflex results in a striking coronary dilation that has two components. The minor component involves a chemoreflex with its efferent pathway in tthe vagi. The major component of coronary dilation follows an increase in the depth of respiration, and its efferent component appears to involve withdrawal of alpha-adrenergic constrictor tone. An almost identical period of reflex coronary dilation followed either forced mechanical or spontaneous hyperinflation in the conscious dog.

Interaction of the chemoreflex and the pulmonary inflation reflex in the regulation of coronary circulation in conscious dogs. The interaction of chemoreflex and pulmonary inflation reflex control of the coronary circulation was examined in conscious dogs by comparing the responses to chemoreflex stimulation (intracarotid injection of nicotine) when ventilation was allowed to increase with those when ventilation was controlled. The responses were also compared with those elicited by both forced mechanical and spontaneous hyperinflation. When the heart rate was constant, intracarotidly administered nicotine induced an increase in the depth of respiration followed closely by an increase in late diastolic coronary flow from 48 +/- 2 to 106 +/- 8 ml/min and a reduction in late diastolic coronary resistance from 1.62 +/- 0.08 to 0.78 +/- 0.06 mm Hg/ml min-1. After beta-receptor and cholinergic blockade, a similar coronary dilation in response to nicotine occurred only when ventilation was allowed to increase. However, when ventilation was controlled, intracarotidly administered nicotine increased coronary resistance after combined beta-receptor and cholinergic blockade. The reflex coronary dilation was not observed after carotid sinus nerve section or after alpha-receptor blockade. Thus, nicotine stimulation of the carotid chemoreflex results in a striking coronary dilation that has two components. The minor component involves a chemoreflex with its efferent pathway in tthe vagi. The major component of coronary dilation follows an increase in the depth of respiration, and its efferent component appears to involve withdrawal of alpha-adrenergic constrictor tone. An almost identical period of reflex coronary dilation followed either forced mechanical or spontaneous hyperinflation in the conscious dog.

pubmed23n0001_102

Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts.

The rate of coronary blood flow was varied in isolated working rat heart preparations to determine its influence on the rate of glocose utilization, tissue high-energy phosphates, and intracellular pH. A 60% reduction in coronary blood flow resulted in a 30% reduction in oxygen consumption, an accelerated rate of glusoe utilization, lower tissue levels of high-energy phosphate, and higher tissue levels of lactate and H+. Ventricular performance deteriorated as reflected by a decrease in heart rate and peak systolic pressure. Further reductions in coronary blood flow resulted in inhibition of glycolysis, a greater decrease in tissue levels of high-energy phosphates, and higher tissue levels of both lactate and H+. These changes in glycolytic flux, tissue metabolites, and ventricular performance were proportional to the degree of restriction in coronary blood flow. The importance of coronary blood flow and washout of the interstitial space in the maintenance of accelerated glycolytic flux in oxygen-deficient hearts is emphasized. It is concluded that acceleration of ATP production from glycolysis can occur only in the marginally ischemic tissue in the peripheral area of tissue supplied by an occluded artery. The central area of tissue which receives a low rate of coronary blood flow will have a reduced rate of ATP production due to both a lack of oxygen and an inhibition of glycolysis.

Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. The rate of coronary blood flow was varied in isolated working rat heart preparations to determine its influence on the rate of glocose utilization, tissue high-energy phosphates, and intracellular pH. A 60% reduction in coronary blood flow resulted in a 30% reduction in oxygen consumption, an accelerated rate of glusoe utilization, lower tissue levels of high-energy phosphate, and higher tissue levels of lactate and H+. Ventricular performance deteriorated as reflected by a decrease in heart rate and peak systolic pressure. Further reductions in coronary blood flow resulted in inhibition of glycolysis, a greater decrease in tissue levels of high-energy phosphates, and higher tissue levels of both lactate and H+. These changes in glycolytic flux, tissue metabolites, and ventricular performance were proportional to the degree of restriction in coronary blood flow. The importance of coronary blood flow and washout of the interstitial space in the maintenance of accelerated glycolytic flux in oxygen-deficient hearts is emphasized. It is concluded that acceleration of ATP production from glycolysis can occur only in the marginally ischemic tissue in the peripheral area of tissue supplied by an occluded artery. The central area of tissue which receives a low rate of coronary blood flow will have a reduced rate of ATP production due to both a lack of oxygen and an inhibition of glycolysis.

pubmed23n0001_103

Mechanisms of glycolytic inhibition in ischemic rat hearts.

The mechanisms of glycolytic inhibition in ischemic myocardium were investigated in the isolated, perfused rat heart. Glycolysis was inhibited at the level of glyceraldehyde-3-phosphate dehydrogenase. The major factors that accounted for the glycolytic inhibition in the ischemic heart compared with the anoxic heart appeared to be higher tissue levels of lactate and H+ in the ischemic tissue. Increased extracellular pH inhibited glycolysis in anoxic and hypoxic hearts much more readily than it did in aerobic hearts. However, maintenance of both extracellular and intracellular pH caused only a modest acceleration of glycolysis in ischemic hearts. Accumulation of tissue lactate and inhibition of glycolysis were directly proportional to the reduction in coronary bloow flow in both anoxic and ischemic hearts. At intracellular lactate concentrations between 15 and 20 mM, glycolysis was inhibited under both conditions. Addition of either 10, 20, or 40 mM lactate to the perfusate inhibited glycolysis in aerobic, anoxic, and ischemic hearts. The effect of lactate did not appear to be mediated through changes in intracellular pH. It is concluded that accumulation of lactate represents a major factor in the inhibition of glycolysis that develops in ischemic hearts.

Mechanisms of glycolytic inhibition in ischemic rat hearts. The mechanisms of glycolytic inhibition in ischemic myocardium were investigated in the isolated, perfused rat heart. Glycolysis was inhibited at the level of glyceraldehyde-3-phosphate dehydrogenase. The major factors that accounted for the glycolytic inhibition in the ischemic heart compared with the anoxic heart appeared to be higher tissue levels of lactate and H+ in the ischemic tissue. Increased extracellular pH inhibited glycolysis in anoxic and hypoxic hearts much more readily than it did in aerobic hearts. However, maintenance of both extracellular and intracellular pH caused only a modest acceleration of glycolysis in ischemic hearts. Accumulation of tissue lactate and inhibition of glycolysis were directly proportional to the reduction in coronary bloow flow in both anoxic and ischemic hearts. At intracellular lactate concentrations between 15 and 20 mM, glycolysis was inhibited under both conditions. Addition of either 10, 20, or 40 mM lactate to the perfusate inhibited glycolysis in aerobic, anoxic, and ischemic hearts. The effect of lactate did not appear to be mediated through changes in intracellular pH. It is concluded that accumulation of lactate represents a major factor in the inhibition of glycolysis that develops in ischemic hearts.

pubmed23n0001_104

Comparison of contractile performance of canine atrial and ventricular muscles.

This study compared the contractile performance of a canine right atrial trabecula with that of a macroscopically indistinguishable trabecula isolated from the right ventricular apex. The heart was removed from nine mongrel puppies weighing 6-8 kg and placed in Krebs-Ringer's bicarbonate solution. The bathing solution contained only 1.25 mmoles of Ca2+ and was bubbled with a 95% O2-5% CO2 gas mixture. Each atrial trabecula was specially selected from the right atrial appendage. Histologically, these trabeculae showed a remarkable longitudinal orientation of the fibers. At Lmax (the length of the muscle at which developed tension was maximum) under identical conditions of temperature, rate of stimulation, ionic milieu, pH, and O2 and CO2 supply, right atrial trabeculae achieved the same developed and total tensions but in a much shorter time than did ventricular trabeculae. In both muscle groups the maximum developed tension averaged about 2.5 g/mm2. Since Lo (expressed as a fraction of Lmax) was less in atrial muscle than it was in ventribular muscle, we concluded that atrial muscle can be stretched considerably more than can ventricular muscle before optimum length is reached. At any given initial muscle length, the maximum of tension rise for atrial trabeculae amounted to at least twice that for ventricular trabeculae. At any given load up to 1.5 g/mm2, the maximum velocity of shortening of an atrial trabecula was about three to four times that of a ventricular trabecula. These results collectively indicate that the contractile performance of the right atrial muscle is in many respects superior to that of the right ventricle, at least under the conditions of these experiments.

Comparison of contractile performance of canine atrial and ventricular muscles. This study compared the contractile performance of a canine right atrial trabecula with that of a macroscopically indistinguishable trabecula isolated from the right ventricular apex. The heart was removed from nine mongrel puppies weighing 6-8 kg and placed in Krebs-Ringer's bicarbonate solution. The bathing solution contained only 1.25 mmoles of Ca2+ and was bubbled with a 95% O2-5% CO2 gas mixture. Each atrial trabecula was specially selected from the right atrial appendage. Histologically, these trabeculae showed a remarkable longitudinal orientation of the fibers. At Lmax (the length of the muscle at which developed tension was maximum) under identical conditions of temperature, rate of stimulation, ionic milieu, pH, and O2 and CO2 supply, right atrial trabeculae achieved the same developed and total tensions but in a much shorter time than did ventricular trabeculae. In both muscle groups the maximum developed tension averaged about 2.5 g/mm2. Since Lo (expressed as a fraction of Lmax) was less in atrial muscle than it was in ventribular muscle, we concluded that atrial muscle can be stretched considerably more than can ventricular muscle before optimum length is reached. At any given initial muscle length, the maximum of tension rise for atrial trabeculae amounted to at least twice that for ventricular trabeculae. At any given load up to 1.5 g/mm2, the maximum velocity of shortening of an atrial trabecula was about three to four times that of a ventricular trabecula. These results collectively indicate that the contractile performance of the right atrial muscle is in many respects superior to that of the right ventricle, at least under the conditions of these experiments.

pubmed23n0001_105

Serum lactate dehydrogenase activity ratios with different substrates.

1. The lactate dehydrogenase activity of 89 sera from patients suffering myocardial infarction and of 55 sera from patients with hepatocellular damage was assayed under optimal conditions using pyruvate, alpha-oxobutyrate, hydroxypyruvate and glyoxylate as substrates. Activity was also measured with lactate as substrate at different pH values. 2. The ratios of activities under these different assay conditions were calculated for both series of patients. Correct differentiations for single ratios ranged from virtually nil for hydroxypyruvate/alpha-oxobutyrate to is greater than 93 per cent for glyoxylate/hydroxypyruvate and glyoxylate/alpha-oxobutyrate. This was little improved by the use of multiple ratios involving up to seven separate assays. 3. The activity ratio of hydroxypyruvate to pyruvate which is consistently greater than unity was found to be inverted in a case of morphine poisoning.

Serum lactate dehydrogenase activity ratios with different substrates. 1. The lactate dehydrogenase activity of 89 sera from patients suffering myocardial infarction and of 55 sera from patients with hepatocellular damage was assayed under optimal conditions using pyruvate, alpha-oxobutyrate, hydroxypyruvate and glyoxylate as substrates. Activity was also measured with lactate as substrate at different pH values. 2. The ratios of activities under these different assay conditions were calculated for both series of patients. Correct differentiations for single ratios ranged from virtually nil for hydroxypyruvate/alpha-oxobutyrate to is greater than 93 per cent for glyoxylate/hydroxypyruvate and glyoxylate/alpha-oxobutyrate. This was little improved by the use of multiple ratios involving up to seven separate assays. 3. The activity ratio of hydroxypyruvate to pyruvate which is consistently greater than unity was found to be inverted in a case of morphine poisoning.

pubmed23n0001_106

Fluorometric assay for N-acetylprocainamide.

We describe a simple, rapid fluorometric assay for separate quantitative analysis of procainamide and N-acetylprocainamide in mixtures. The effective lenear range (fluorescence vs. concentration) in serum is 0.1 to 10.0 mg/liter, regardless of the ratio (by weight) of the two drugs from 1:10 to 10:1. Analytical recoveries by the extraction method used were 100.0 +/- 3.0% and 98.0 +/- 4.0%, respectively. For determination of either compound, the maximum coefficient of variation was 10%.

Fluorometric assay for N-acetylprocainamide. We describe a simple, rapid fluorometric assay for separate quantitative analysis of procainamide and N-acetylprocainamide in mixtures. The effective lenear range (fluorescence vs. concentration) in serum is 0.1 to 10.0 mg/liter, regardless of the ratio (by weight) of the two drugs from 1:10 to 10:1. Analytical recoveries by the extraction method used were 100.0 +/- 3.0% and 98.0 +/- 4.0%, respectively. For determination of either compound, the maximum coefficient of variation was 10%.

pubmed23n0001_107

Rapid kinetic measurement of lactate in plasma with a centrifugal analyzer.

In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 mul of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.

Rapid kinetic measurement of lactate in plasma with a centrifugal analyzer. In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 mul of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.

pubmed23n0001_108

Improved high-resolution high-voltage paper electrophoresis system for use in screening for aminoacidopathies.

High-voltage paper electrophoresis of small samples of serum and urine at pH 6.0 resolves basic and acidic amino acids and separates them from the neutral amino acids. For separation and identification of the neutral amino acids, the appropriate area of the electrophoretogram is cut out, sewn onto a second sheet of paper, and rerun at pH 1.9. By this method, amino acids are rapidly resolved. It is suited for use with special procedures such as oxidation of biological fluid with performic acid and specific staining for confirmation of amino acid identification.

Improved high-resolution high-voltage paper electrophoresis system for use in screening for aminoacidopathies. High-voltage paper electrophoresis of small samples of serum and urine at pH 6.0 resolves basic and acidic amino acids and separates them from the neutral amino acids. For separation and identification of the neutral amino acids, the appropriate area of the electrophoretogram is cut out, sewn onto a second sheet of paper, and rerun at pH 1.9. By this method, amino acids are rapidly resolved. It is suited for use with special procedures such as oxidation of biological fluid with performic acid and specific staining for confirmation of amino acid identification.

pubmed23n0001_109

A clinical method for the determination of serum gamma-glutamyl transpeptidase.

A simple, highly sensitive and reproducible method for the assay of gamma-glutamyl transpeptidase (EC 2.3.2-) activity is introduced, using gamma-glutamyl-p-nitroanilide as a substrate and glycylglycine as an acceptor in 50 g/l of polyoxyethylene nonylphenol. Serum transpeptidase activity was assayed in 1080 healthy adults, the normal mean value being 14.8 mU/ml. The diagnostic evaluation of the enzyme in various hepatobiliary diseases is also discussed.

A clinical method for the determination of serum gamma-glutamyl transpeptidase. A simple, highly sensitive and reproducible method for the assay of gamma-glutamyl transpeptidase (EC 2.3.2-) activity is introduced, using gamma-glutamyl-p-nitroanilide as a substrate and glycylglycine as an acceptor in 50 g/l of polyoxyethylene nonylphenol. Serum transpeptidase activity was assayed in 1080 healthy adults, the normal mean value being 14.8 mU/ml. The diagnostic evaluation of the enzyme in various hepatobiliary diseases is also discussed.

pubmed23n0001_110

Treatment of renal hypertension.

There are different types of renal hypertension: hypertension due to parenchymal renal disease, renovascular hypertension, hypertension due to urological disease, hypertension of endstage renal disease. Treatment has to consider-above all-the possibility of specific, medical or surgical procedures that may cause the underlying condition. If the underlying disease is not amenable to specific therapy, symptomatic medical treatment to lower blood pressure is indicated: besides control of sodium-intake and body weight antihypertensive drugs are generally indicated. We use them, alone or in combination, in the following line of order: diuretics, beta-adrenergic blockers, dihydralazine, reserpine, clonidine, alpha-methyldopa, guanethidine.

Treatment of renal hypertension. There are different types of renal hypertension: hypertension due to parenchymal renal disease, renovascular hypertension, hypertension due to urological disease, hypertension of endstage renal disease. Treatment has to consider-above all-the possibility of specific, medical or surgical procedures that may cause the underlying condition. If the underlying disease is not amenable to specific therapy, symptomatic medical treatment to lower blood pressure is indicated: besides control of sodium-intake and body weight antihypertensive drugs are generally indicated. We use them, alone or in combination, in the following line of order: diuretics, beta-adrenergic blockers, dihydralazine, reserpine, clonidine, alpha-methyldopa, guanethidine.

pubmed23n0001_111

Anaerobic glycolysis in normal human erythrocytes incubated in vitro with sodium salicylate.

1. Some effects of sodium salicylate upon anaerobic glycolysis have been studied in normal human erythrocytes incubated for up to 6 h at 37 degrees C in autologous sera. 2. Both glucose consumption and lactate production were stimulated by concentrations of salicylate up to 60 mmol/l but at the highest concentration used (90 mmol/l) an initial stimulus was followed by inhibition of glycolysis. 3. Losses occurred of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-phosphate(AMP)at higher concentrations of salicylate and there was a concomitant increase of inorganic phosphate. 4. Other phosphate esters underwent concentration changes at higher concentrations of salicylate that reflected inadequate concentrations of ATP for glycolysis. 5. The rates of sodium efflux from, and potassium influx into, erythrocytes were unaffected by the presence of salicylate at concentrations sufficient to stimulate glycolysis.

Anaerobic glycolysis in normal human erythrocytes incubated in vitro with sodium salicylate. 1. Some effects of sodium salicylate upon anaerobic glycolysis have been studied in normal human erythrocytes incubated for up to 6 h at 37 degrees C in autologous sera. 2. Both glucose consumption and lactate production were stimulated by concentrations of salicylate up to 60 mmol/l but at the highest concentration used (90 mmol/l) an initial stimulus was followed by inhibition of glycolysis. 3. Losses occurred of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-phosphate(AMP)at higher concentrations of salicylate and there was a concomitant increase of inorganic phosphate. 4. Other phosphate esters underwent concentration changes at higher concentrations of salicylate that reflected inadequate concentrations of ATP for glycolysis. 5. The rates of sodium efflux from, and potassium influx into, erythrocytes were unaffected by the presence of salicylate at concentrations sufficient to stimulate glycolysis.

pubmed23n0001_112

Arterial catecholamines in hypoxic exercise in man.

1. We measured the minute ventilation and arterial blood catecholamine concentrations in four normal men standing and at two levels of moderate treadmill exercise breathing 14% oxygen or air. 2. Minute ventilation was significantly higher during hypoxic exercise than during normoxic exercise at an oxygen uptake of 1500 ml/min. 3. Arterial plasma noradrenaline during hypoxic exercise at an oxygen uptake of 1500 ml/min was significantly greater than at rest. 4. Arterial plasma noradrenaline during normoxic exercise at an oxygen uptake of 1500 ml/min was not elevated above the resting concentration. 5. The results are compatible with the suggestion that increased concentrations of arterial plasma noradrenaline contribute to the hypoxic potentiation of the respiratory response to moderate exercise.

Arterial catecholamines in hypoxic exercise in man. 1. We measured the minute ventilation and arterial blood catecholamine concentrations in four normal men standing and at two levels of moderate treadmill exercise breathing 14% oxygen or air. 2. Minute ventilation was significantly higher during hypoxic exercise than during normoxic exercise at an oxygen uptake of 1500 ml/min. 3. Arterial plasma noradrenaline during hypoxic exercise at an oxygen uptake of 1500 ml/min was significantly greater than at rest. 4. Arterial plasma noradrenaline during normoxic exercise at an oxygen uptake of 1500 ml/min was not elevated above the resting concentration. 5. The results are compatible with the suggestion that increased concentrations of arterial plasma noradrenaline contribute to the hypoxic potentiation of the respiratory response to moderate exercise.

pubmed23n0001_113

Effects of graded infusions of monomethylmethacrylate on coagulation, blood lipids, respiration and circulation. An experimental study in dogs.

In 4 dogs injected intravenously (i.v.) with 125I labeled fibrinogen, 51Cr labeled platelets and 99mTc labeled albumin, and subjected to successively increasing amounts of i.v. infused monomethylmethacrylate, doses corresponding to the amounts released into the blood stream following implantation of acrylic cement during total hip replacements did not affect the clotting mechanism, did not cause trapping of platelets and fibrin in the lungs, did not generate fat emboli, and did not cause depression of the arterial oxygen tension or blood pressure. Monomethylmethacrylate in whole blood was associated with both blood cells and plasma.

Effects of graded infusions of monomethylmethacrylate on coagulation, blood lipids, respiration and circulation. An experimental study in dogs. In 4 dogs injected intravenously (i.v.) with 125I labeled fibrinogen, 51Cr labeled platelets and 99mTc labeled albumin, and subjected to successively increasing amounts of i.v. infused monomethylmethacrylate, doses corresponding to the amounts released into the blood stream following implantation of acrylic cement during total hip replacements did not affect the clotting mechanism, did not cause trapping of platelets and fibrin in the lungs, did not generate fat emboli, and did not cause depression of the arterial oxygen tension or blood pressure. Monomethylmethacrylate in whole blood was associated with both blood cells and plasma.

pubmed23n0001_114

Influence of pH and hypoxia on the success of defibrillation.

Clinical impressions about the problem of defibrillation during states of acid-base imbalance and hypoxia have been influenced by studies involving the effect of these derangements on the ventricular fibrillation threshold. Based on body weight, energy requirements for defibrillation in normal dogs were compared to requirements in dogs subjected to commonly encountered acid-base disturbances and severe hypoxemia. No significant differences were found. Seventy-five percent of all animals in the study were electrically converted with low-to-moderate levels of energy. The incidence of spontaneous resumption of circulation following defibrillation was lowest in animals subjected to metabolic acidosis and hypoxia. The results suggest that pH and blood gas alterations, previously shown to influence the normal ventricular fibrillation threshold, do not significantly affect the normal defibrillation threshold.

Influence of pH and hypoxia on the success of defibrillation. Clinical impressions about the problem of defibrillation during states of acid-base imbalance and hypoxia have been influenced by studies involving the effect of these derangements on the ventricular fibrillation threshold. Based on body weight, energy requirements for defibrillation in normal dogs were compared to requirements in dogs subjected to commonly encountered acid-base disturbances and severe hypoxemia. No significant differences were found. Seventy-five percent of all animals in the study were electrically converted with low-to-moderate levels of energy. The incidence of spontaneous resumption of circulation following defibrillation was lowest in animals subjected to metabolic acidosis and hypoxia. The results suggest that pH and blood gas alterations, previously shown to influence the normal ventricular fibrillation threshold, do not significantly affect the normal defibrillation threshold.

pubmed23n0001_115

Pleural effusion associated with aortitis syndrome.

A patient with aortitis syndrome had a pleural effusion which subsided but reappeared with an exacerbation of aortitis symptoms while under antituberculosis treatment. The character of the fluid was that of an exudate, and the glucose concentration was normal. Clinical and laboratory features of the case suggest that the effusion was part of the aortitis syndrome per se.

Pleural effusion associated with aortitis syndrome. A patient with aortitis syndrome had a pleural effusion which subsided but reappeared with an exacerbation of aortitis symptoms while under antituberculosis treatment. The character of the fluid was that of an exudate, and the glucose concentration was normal. Clinical and laboratory features of the case suggest that the effusion was part of the aortitis syndrome per se.

pubmed23n0001_116

[Transplantation and preservation of tissue-typized skin in burns].

At the Department of Plastic Surgery and Burns, Berufsgenossenschaftliche Unfalklinik Ludwigshafen-Oggersheim, a skinbank with typed skin has been established. The storage system consists of deep-freezing at -196 degrees C in liquid nitrogen. The transplantation of HL-A typed skin was evaluated from the clinical and histological point of view. Due to the effort necessary for such an undertaking this method is critically reviewed.

[Transplantation and preservation of tissue-typized skin in burns]. At the Department of Plastic Surgery and Burns, Berufsgenossenschaftliche Unfalklinik Ludwigshafen-Oggersheim, a skinbank with typed skin has been established. The storage system consists of deep-freezing at -196 degrees C in liquid nitrogen. The transplantation of HL-A typed skin was evaluated from the clinical and histological point of view. Due to the effort necessary for such an undertaking this method is critically reviewed.

pubmed23n0001_117

Charge properties of human pituitary and amniotic fluid prolactins.

The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The "large" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.

Charge properties of human pituitary and amniotic fluid prolactins. The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The "large" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.

pubmed23n0001_118

Human and monkey prolactin and growth hormone: separation of polymorphic forms by isoelectric focusing.

The feasibility of using isoelectric focusing for the separation of primate pituitary growth hormone from prolactin and for the characterization of polymorphic forms of these hormones was explored. In a pH 3--10 gradient, extracts of both human and cynomolgus monkey pituitaries were each resolved into 4 growth hormone components and at least 3 prolactin components, as shown by radioimmunoassay. In narrower gradients (of 2--3 pH units) greater resolution was achieved; the principal growth hormone components were well separated from the principal prolactin components but there was overlapping of some minor components. A partially purified human prolactin preparation was found to contain 4 prolactin components, one of which had a prolactin/growth hormone ratio of 760. Clinical grade human growth hormone was also resolved into at least 5 prolactin and 5 growth hormone components, many of which had higher pI values than those found in pituitary extract. Under the conditions used, both growth hormone and prolactin were found to be polymorphic with respect to isoelectric point. Some of the human prolactin components were found to contain less than 0.2% growth hormone by radioimmunoassay. Monkey growth hormone containing 0.01% prolactin was isolated. These findings demonstrate that isoelectric focusing is useful for the preparation of both growth hormone and prolactin which are essentially free of one another. Furthermore, the polymorphic forms were repeatedly found in preparations obtained by several methods and from 2 different species, suggesting that these forms are not artifacts.

Human and monkey prolactin and growth hormone: separation of polymorphic forms by isoelectric focusing. The feasibility of using isoelectric focusing for the separation of primate pituitary growth hormone from prolactin and for the characterization of polymorphic forms of these hormones was explored. In a pH 3--10 gradient, extracts of both human and cynomolgus monkey pituitaries were each resolved into 4 growth hormone components and at least 3 prolactin components, as shown by radioimmunoassay. In narrower gradients (of 2--3 pH units) greater resolution was achieved; the principal growth hormone components were well separated from the principal prolactin components but there was overlapping of some minor components. A partially purified human prolactin preparation was found to contain 4 prolactin components, one of which had a prolactin/growth hormone ratio of 760. Clinical grade human growth hormone was also resolved into at least 5 prolactin and 5 growth hormone components, many of which had higher pI values than those found in pituitary extract. Under the conditions used, both growth hormone and prolactin were found to be polymorphic with respect to isoelectric point. Some of the human prolactin components were found to contain less than 0.2% growth hormone by radioimmunoassay. Monkey growth hormone containing 0.01% prolactin was isolated. These findings demonstrate that isoelectric focusing is useful for the preparation of both growth hormone and prolactin which are essentially free of one another. Furthermore, the polymorphic forms were repeatedly found in preparations obtained by several methods and from 2 different species, suggesting that these forms are not artifacts.

pubmed23n0001_119

Ecological observation of the 137Cs-contamination in beef of animals from the southern-Bavarian area.

Certain climatic and edaphic conformations in the Bavarian sub-alpine mountains and in the Alps favor above all the development of a land utilization system and farm structures similar to those in the northern part of Scandinavia. In 1963/64, the years of the highest environmental contamination up to the present, we established in 600 beef samples from the round or shoulder of male and female cattle (mainly Highland cattle) close connections between the 137Cs-contamination of green crop and the long lastnig yearly precipitation quantities, as well as certain relations between the 137Cs-contamination of meat and differences in the feeding and keeping of the animals. During summer-seasons (April-October), beef of cattle from pastures with heavy rainfall (Alps) was contaiminated by 137Cs up to 15 times more than that of confined animals. Hereby the rate of 137Cs-contamination in the meat of grazing cattle was nearly proportional to the quantities of precipitation. When confined cattle were fed on pastures in autumn after harvesting for 2 to 3 weeks, a quick increase of 137Cs-contamination of the meat was caused within this time up to values which in this district were otherwise only observed in grazing cattle. The lower 137Cs-content in meat of cattle housed during the summer season is due to the more varied fodder, which is at that time less contaminated than green crop. During the winter season (November to March), the highest contaminations in the meat of confined (Bohemian Forest) or grazing cattle (Alps) was measured when the animals in these districts were almost exclusively fed with fodder from the own farmground or with leafy silage. The highest contamination was almost regularly noticed in January, February and March, as generally during these months the highly contaminated first cut hay is fed. Here the meat was often even more contaminated than that of grazing cattle. After the quick decrease of 137Cs in fallout noticed in the years 1964 and 1965, in 1965/66 a dependance in the 137Cs-contamination of beef on the methods of keeping and feeding could still be observed in only the extreme cases (Alps, Bavarian- and Bohemian Forest); though in general, meat of animals from districts with heavy rainfall was slightly more contaminated than meat of animals from regions with less precipitation.

Ecological observation of the 137Cs-contamination in beef of animals from the southern-Bavarian area. Certain climatic and edaphic conformations in the Bavarian sub-alpine mountains and in the Alps favor above all the development of a land utilization system and farm structures similar to those in the northern part of Scandinavia. In 1963/64, the years of the highest environmental contamination up to the present, we established in 600 beef samples from the round or shoulder of male and female cattle (mainly Highland cattle) close connections between the 137Cs-contamination of green crop and the long lastnig yearly precipitation quantities, as well as certain relations between the 137Cs-contamination of meat and differences in the feeding and keeping of the animals. During summer-seasons (April-October), beef of cattle from pastures with heavy rainfall (Alps) was contaiminated by 137Cs up to 15 times more than that of confined animals. Hereby the rate of 137Cs-contamination in the meat of grazing cattle was nearly proportional to the quantities of precipitation. When confined cattle were fed on pastures in autumn after harvesting for 2 to 3 weeks, a quick increase of 137Cs-contamination of the meat was caused within this time up to values which in this district were otherwise only observed in grazing cattle. The lower 137Cs-content in meat of cattle housed during the summer season is due to the more varied fodder, which is at that time less contaminated than green crop. During the winter season (November to March), the highest contaminations in the meat of confined (Bohemian Forest) or grazing cattle (Alps) was measured when the animals in these districts were almost exclusively fed with fodder from the own farmground or with leafy silage. The highest contamination was almost regularly noticed in January, February and March, as generally during these months the highly contaminated first cut hay is fed. Here the meat was often even more contaminated than that of grazing cattle. After the quick decrease of 137Cs in fallout noticed in the years 1964 and 1965, in 1965/66 a dependance in the 137Cs-contamination of beef on the methods of keeping and feeding could still be observed in only the extreme cases (Alps, Bavarian- and Bohemian Forest); though in general, meat of animals from districts with heavy rainfall was slightly more contaminated than meat of animals from regions with less precipitation.

pubmed23n0001_120

Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase.

Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.

Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase. Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.

pubmed23n0001_121

Biochemical aspects of renal ammonia formation in metabolic acidosis.

In omnivorous creatures, the diet is acidogenic, especially as a result of the meat content, which gives rise to phosphoric and sulphuric acids, i.e., to metabolic acidosis. In the short term, metabolic acids are buffered by tissue proteins and bicarbonate (the 'alkali reserve'). In the longer term, acid must be excreted, or neutralized with base which is also generated from the diet, by conversion of dietary amino-nitrogen to ammonia. The final steps of this process occur in the kidney, which converts circulating glutamine to ammonia, and to carbon products such as glucose and carbon dioxide, by metabolic reactions which adapt during acidosis to generate more ammonia and maintain an increased renal ammonia content. The complex mechanisms which govern the formation of ammonia, glucose and carbon dioxide from glutamine, involving the reactions of amino acids, the tricarboxylic acid cycle, and gluconeogenesis, are reviewed.

Biochemical aspects of renal ammonia formation in metabolic acidosis. In omnivorous creatures, the diet is acidogenic, especially as a result of the meat content, which gives rise to phosphoric and sulphuric acids, i.e., to metabolic acidosis. In the short term, metabolic acids are buffered by tissue proteins and bicarbonate (the 'alkali reserve'). In the longer term, acid must be excreted, or neutralized with base which is also generated from the diet, by conversion of dietary amino-nitrogen to ammonia. The final steps of this process occur in the kidney, which converts circulating glutamine to ammonia, and to carbon products such as glucose and carbon dioxide, by metabolic reactions which adapt during acidosis to generate more ammonia and maintain an increased renal ammonia content. The complex mechanisms which govern the formation of ammonia, glucose and carbon dioxide from glutamine, involving the reactions of amino acids, the tricarboxylic acid cycle, and gluconeogenesis, are reviewed.

pubmed23n0001_122

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. I. Experimental extrahepatic cholestasis in rabbits.

Serum, liver and renal gamma-glutamyl transpeptidase (GGT) activities were studied in four groups of rabbits: controls, rabbits with obstructive extrahepatic cholestasis, rabbits with obstructive anuria, and animals with combined obstructive extrahepatic cholestasis and obstructive anuria. Serum GGT was essentially increased in rabbits with obstructive extrahepatic cholestasis, showing peak values in the combined cholestasis + obstructive anuria group, and practically normal values in animals with anuria. Liver GGT was increased in both cholestasis groups, but the increase was less prominent than the increase in serum GGT and there was no correlation between them. In both anuric groups renal GGT was reduced, probably as a result of inhibited enzyme synthesis secondary to the altered conditions for adequate renal function. The results obtained are suggestive of a probable renal involvement in the formation of the serum GGT activity level.

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. I. Experimental extrahepatic cholestasis in rabbits. Serum, liver and renal gamma-glutamyl transpeptidase (GGT) activities were studied in four groups of rabbits: controls, rabbits with obstructive extrahepatic cholestasis, rabbits with obstructive anuria, and animals with combined obstructive extrahepatic cholestasis and obstructive anuria. Serum GGT was essentially increased in rabbits with obstructive extrahepatic cholestasis, showing peak values in the combined cholestasis + obstructive anuria group, and practically normal values in animals with anuria. Liver GGT was increased in both cholestasis groups, but the increase was less prominent than the increase in serum GGT and there was no correlation between them. In both anuric groups renal GGT was reduced, probably as a result of inhibited enzyme synthesis secondary to the altered conditions for adequate renal function. The results obtained are suggestive of a probable renal involvement in the formation of the serum GGT activity level.

pubmed23n0001_123

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. II. Experimental hexachlorobenzene porphyria in rabbits.

The mechanism responsible for the changes in serum and liver gamma-glutamyl transpeptidase (gamma-GT) activity was studied in a model of experimental hexachlorobenzene porphyria in rabbits. Porphyria followed the administration of hexachlorobenzene in doses of 280 mumol - kg-1 body weight, which were given daily through a gastric tube over a 20-day period. Serum gamma-GT activity and the activities of the lysosomal enzymes beta-N-acetylglucosaminidase and alpha-mannosidase were increased, whereas L-aspartate: 2-oxoglutarate aminotransferase and L-alanine: 2-oxoglutarate aminotransferase and L-alanine: 2-oxoglutarate aminotransferase remained unaltered. There was a considerable increase in liver microsomal protein, gamma-GT, cytochrome P-450, anilinehydroxylase, aminopyrine-demethylase and delta-aminolevulinic acid synthase. In the liver gamma-GT was detected in the microsomes as well as in the cytoplasm where enzymatic activity was higher. The high correlation coefficient between liver gamma-GT, cytochrome P-450 and delta-aminolevulinic acid synthase witnesses a hexachlorobenzene-induced gamma-GT formation in the liver. A statistically significant correlation between serum and liver gamma-GT activity was also found. These data strongly suggest that the increase in serum gamma-GT activity may result from the induction of the enzyme in the liver.

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. II. Experimental hexachlorobenzene porphyria in rabbits. The mechanism responsible for the changes in serum and liver gamma-glutamyl transpeptidase (gamma-GT) activity was studied in a model of experimental hexachlorobenzene porphyria in rabbits. Porphyria followed the administration of hexachlorobenzene in doses of 280 mumol - kg-1 body weight, which were given daily through a gastric tube over a 20-day period. Serum gamma-GT activity and the activities of the lysosomal enzymes beta-N-acetylglucosaminidase and alpha-mannosidase were increased, whereas L-aspartate: 2-oxoglutarate aminotransferase and L-alanine: 2-oxoglutarate aminotransferase and L-alanine: 2-oxoglutarate aminotransferase remained unaltered. There was a considerable increase in liver microsomal protein, gamma-GT, cytochrome P-450, anilinehydroxylase, aminopyrine-demethylase and delta-aminolevulinic acid synthase. In the liver gamma-GT was detected in the microsomes as well as in the cytoplasm where enzymatic activity was higher. The high correlation coefficient between liver gamma-GT, cytochrome P-450 and delta-aminolevulinic acid synthase witnesses a hexachlorobenzene-induced gamma-GT formation in the liver. A statistically significant correlation between serum and liver gamma-GT activity was also found. These data strongly suggest that the increase in serum gamma-GT activity may result from the induction of the enzyme in the liver.

pubmed23n0001_124

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise. 15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.

pubmed23n0001_125

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide.

1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide. 1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.

pubmed23n0001_126

Identification of chloride-binding sites in hemoglobin by nuclear-magnetic-resonance quadrupole-relaxation studies of hemoglobin digests.

35Cl minus-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl minus NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-beta146-Asp-beta94 and at or near Val-alpha1-Arg-alpha141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.

Identification of chloride-binding sites in hemoglobin by nuclear-magnetic-resonance quadrupole-relaxation studies of hemoglobin digests. 35Cl minus-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl minus NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-beta146-Asp-beta94 and at or near Val-alpha1-Arg-alpha141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.

pubmed23n0001_127

The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhibitor, in growing Clostridium pasteurianum.

1. The intracellular pH was measured in growing Clostridium pasteurianum with and acid-base equilibrium distribution method. [14C]Dimethyloxazolidinedione, [14]methylamine and [14C]acetic acid were used as "deltapH-indicators". During growth the extracellular pH decreased from 7.1 to 5.1; simultaneously the intracellular pH changed from 7.5 to 5.9. Thus, the intracellular pH was more alkaline than the extracellular pH by 0.4 to 0.8 pH-units. 2. This pH gradient (interior alkaline) was abolished by the proton conductor carbonylcyanide m-chlorophenylhydrazone and the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The pH gradient could not be demonstrated in cells depleted of an energy substrate. These results suggest that the pH gradient is formed by an ATPase-driven extrusion of protons from the cells rather than by a Donnan potential. 3. Growth of the organism was inhibited by low concentrations of both carbonylcyanide m-chlorophenylhydrazone (5 muM) and dicyclohexylcarbodiimide (5 muM). This finding suggests that the pH gradient is essential for the growing cell as it may be required for substrate accumulation and other types of transport processes.

The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhibitor, in growing Clostridium pasteurianum. 1. The intracellular pH was measured in growing Clostridium pasteurianum with and acid-base equilibrium distribution method. [14C]Dimethyloxazolidinedione, [14]methylamine and [14C]acetic acid were used as "deltapH-indicators". During growth the extracellular pH decreased from 7.1 to 5.1; simultaneously the intracellular pH changed from 7.5 to 5.9. Thus, the intracellular pH was more alkaline than the extracellular pH by 0.4 to 0.8 pH-units. 2. This pH gradient (interior alkaline) was abolished by the proton conductor carbonylcyanide m-chlorophenylhydrazone and the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The pH gradient could not be demonstrated in cells depleted of an energy substrate. These results suggest that the pH gradient is formed by an ATPase-driven extrusion of protons from the cells rather than by a Donnan potential. 3. Growth of the organism was inhibited by low concentrations of both carbonylcyanide m-chlorophenylhydrazone (5 muM) and dicyclohexylcarbodiimide (5 muM). This finding suggests that the pH gradient is essential for the growing cell as it may be required for substrate accumulation and other types of transport processes.

pubmed23n0001_128

D-alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis.

Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki - 4 X 10(-9) M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 X 10(7) M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD-carboxtpeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. mirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.

D-alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki - 4 X 10(-9) M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 X 10(7) M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD-carboxtpeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. mirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.

pubmed23n0001_129

Pigeon-liver NAD kinase. The structural and kinetic basis of regulation of NADPH.

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.

Pigeon-liver NAD kinase. The structural and kinetic basis of regulation of NADPH. NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.

pubmed23n0001_130

Hydrogen ion changes and contractile behavior in the perfused rat heart.

The effect of acid-base alterations was analyzed using isolated rat hearts perfused at constant coronary perfusion pressure, and stimulated to contract at constant rat. The amount of shortening in the major axis and its derivative were measured to assess myocardial contractility. Both the 'respiratory' and 'metabolic' alterations affected the contractile behavior to the same extent. In the physiological range studied by us, acidosis depresses and alkalosis increases myocardial contraction. However acidosis seems to depress contractility more than the enhancement produced by the same change in pH towards the alkalotic side. When either amount of shortening or max dl/dt was plotted as a function of hydrogen ion acitvity (aH+) a linear correlation was obtained, either with pure 'metabolic' or 'respiratory' acid-base induced alterations (correlation coefficients higher than -.95; P less than .01). Our findings suggest that in the range studied by us, contraction of the perfused rat heart following acid-base alterations, is a linear function of hydrogen ion activity.

Hydrogen ion changes and contractile behavior in the perfused rat heart. The effect of acid-base alterations was analyzed using isolated rat hearts perfused at constant coronary perfusion pressure, and stimulated to contract at constant rat. The amount of shortening in the major axis and its derivative were measured to assess myocardial contractility. Both the 'respiratory' and 'metabolic' alterations affected the contractile behavior to the same extent. In the physiological range studied by us, acidosis depresses and alkalosis increases myocardial contraction. However acidosis seems to depress contractility more than the enhancement produced by the same change in pH towards the alkalotic side. When either amount of shortening or max dl/dt was plotted as a function of hydrogen ion acitvity (aH+) a linear correlation was obtained, either with pure 'metabolic' or 'respiratory' acid-base induced alterations (correlation coefficients higher than -.95; P less than .01). Our findings suggest that in the range studied by us, contraction of the perfused rat heart following acid-base alterations, is a linear function of hydrogen ion activity.

pubmed23n0001_131

Changes of EEG and pulmonary venous admixture during a protein load in patients with cirrhosis.

21 patients with cirrhosis of the liver and 24 control patients were studied before and after a protein load (120 g protein per day during one week). An EEG was recorded and a visual assessment of frequency pattern was performed. Venous admixture was estimated during hyperoxia. According to the EEG frequency pattern the patient group with cirrhosis was subdivided into those with EEG slowing after the protein load (n = 7) and those without (n = 14). The following results were obtained: 1) Resting arterial blood gases did not change in either group. 2) There was a significant increase of the AaD02 (difference between alveolar p02 and peripheral arterial p02) in cirrhotics and controls. 3) The increase in AaD02 was significantly larger in those cirrhotics showing EEG slowing compared to those without EEG - slowing or to the controls. 4) Fractional venous admisture increased significantly in those cirrhotics showing EEG slowing. There was no significant change in those patients who did not show EEG changes or in the controls.

Changes of EEG and pulmonary venous admixture during a protein load in patients with cirrhosis. 21 patients with cirrhosis of the liver and 24 control patients were studied before and after a protein load (120 g protein per day during one week). An EEG was recorded and a visual assessment of frequency pattern was performed. Venous admixture was estimated during hyperoxia. According to the EEG frequency pattern the patient group with cirrhosis was subdivided into those with EEG slowing after the protein load (n = 7) and those without (n = 14). The following results were obtained: 1) Resting arterial blood gases did not change in either group. 2) There was a significant increase of the AaD02 (difference between alveolar p02 and peripheral arterial p02) in cirrhotics and controls. 3) The increase in AaD02 was significantly larger in those cirrhotics showing EEG slowing compared to those without EEG - slowing or to the controls. 4) Fractional venous admisture increased significantly in those cirrhotics showing EEG slowing. There was no significant change in those patients who did not show EEG changes or in the controls.

pubmed23n0001_132

An evaluation of factors affecting the in vitro bioassay for erythropoietin.

Two main aspects of the in vitro mouse foetal liver cell assay for Erythroid Stimulating Factor (ESF) in human sera have been investigated. The haem extraction process has been shown to give specific and quantitative recovery of 59Fe labelled haem from haemoglobin thus confirming that the material assayed in human sera is stimulating the synthesis of this protein. The extraction procedure can be simplified considerably by prior mixing of the reagents without significantly influencing the results. Several serum constituents (citrate, testosterone, B12, folic acid and iron) have been investigated over a range of concentrations for possible effects on the cultures. Generally only small effects on haem synthesis were observed. It is concluded that variations in the levels of these factors in sera from treated patients will not produce any significant alterations in the estimated ESF concentrations.

An evaluation of factors affecting the in vitro bioassay for erythropoietin. Two main aspects of the in vitro mouse foetal liver cell assay for Erythroid Stimulating Factor (ESF) in human sera have been investigated. The haem extraction process has been shown to give specific and quantitative recovery of 59Fe labelled haem from haemoglobin thus confirming that the material assayed in human sera is stimulating the synthesis of this protein. The extraction procedure can be simplified considerably by prior mixing of the reagents without significantly influencing the results. Several serum constituents (citrate, testosterone, B12, folic acid and iron) have been investigated over a range of concentrations for possible effects on the cultures. Generally only small effects on haem synthesis were observed. It is concluded that variations in the levels of these factors in sera from treated patients will not produce any significant alterations in the estimated ESF concentrations.

pubmed23n0001_133

[Synthesis of N-substituted isoindolines].

Some derivatives of isoindoline were prepared in order to test their cardiovascular activity. Pharmacological tests showed that some of the compounds had moderate alpha-blocking and coronarodilatory activity whereas others had some local anesthetic activity.

[Synthesis of N-substituted isoindolines]. Some derivatives of isoindoline were prepared in order to test their cardiovascular activity. Pharmacological tests showed that some of the compounds had moderate alpha-blocking and coronarodilatory activity whereas others had some local anesthetic activity.

pubmed23n0001_134

[Mechanisms of the thermogenic action of noradrenaline during adaptation to cold].

In white rats both adapted and unadapted to cold, the RQ dynamics during cold exposure, noradrenaline and ganglion blocking agent administration, were studied. The adapted animals' RQ in thermoneutral conditions was shown to be a little higher than in the control rats; 0.5 mg/kg noradrenaline injections induced a clear RQ decrease in the former and did not influence the latters' RQ. Cold exposure was followed by a RQ decrease in both. Ganglion blocking agent administration decreased the RQ in the adapted animals and prevented it from falling in the control those. Noradrenaline is supposed to be the main but not the only factor activating lipolysis in the cold adapted animals.

[Mechanisms of the thermogenic action of noradrenaline during adaptation to cold]. In white rats both adapted and unadapted to cold, the RQ dynamics during cold exposure, noradrenaline and ganglion blocking agent administration, were studied. The adapted animals' RQ in thermoneutral conditions was shown to be a little higher than in the control rats; 0.5 mg/kg noradrenaline injections induced a clear RQ decrease in the former and did not influence the latters' RQ. Cold exposure was followed by a RQ decrease in both. Ganglion blocking agent administration decreased the RQ in the adapted animals and prevented it from falling in the control those. Noradrenaline is supposed to be the main but not the only factor activating lipolysis in the cold adapted animals.

pubmed23n0001_135

Chromosomal basis of the merozygosity in a partially deploid mutant of Pneumococcus.

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

Chromosomal basis of the merozygosity in a partially deploid mutant of Pneumococcus. A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

pubmed23n0001_136

Recombination as a requirement for segregation of a partially diploid mutant of Pneumococcus.

Conditions are described in which the pneumococcal mutant strain sulr-c, resistant to the drug sulfanilamide, gives rise to sensitive segregants resistant to nitrobenzoic acid at a frequency constant with time. This segregant frequency is markedly enhanced upon exposure of the cells to doses of ultraviolet light or mitomycin C that permit survival of 50% to 90% of the cells. Treatment with acridine orange diminishes the segregant frequency. From the known influences of these three agents on genetic recombination, we propose that a recombination event is necessary in the generation of segregants.--During a period of incubation following treatment with ultraviolet light or mitomycin C, cell division resumes and the original segregant frequency is restored. Thus potential segregants are either unable to replicate in the absence of selection, or they are under-represented among the cells dividing soon after treatment.--If the sulr-c mutation is introduced into a mutant pneumococcal strain lacking an ATP-dependent exonuclease activity and deficient in recombination with transforming DNA, segregant frequencies are unaffected. This fact may indicate limits upon the type of recombination event responsible for segregation.

Recombination as a requirement for segregation of a partially diploid mutant of Pneumococcus. Conditions are described in which the pneumococcal mutant strain sulr-c, resistant to the drug sulfanilamide, gives rise to sensitive segregants resistant to nitrobenzoic acid at a frequency constant with time. This segregant frequency is markedly enhanced upon exposure of the cells to doses of ultraviolet light or mitomycin C that permit survival of 50% to 90% of the cells. Treatment with acridine orange diminishes the segregant frequency. From the known influences of these three agents on genetic recombination, we propose that a recombination event is necessary in the generation of segregants.--During a period of incubation following treatment with ultraviolet light or mitomycin C, cell division resumes and the original segregant frequency is restored. Thus potential segregants are either unable to replicate in the absence of selection, or they are under-represented among the cells dividing soon after treatment.--If the sulr-c mutation is introduced into a mutant pneumococcal strain lacking an ATP-dependent exonuclease activity and deficient in recombination with transforming DNA, segregant frequencies are unaffected. This fact may indicate limits upon the type of recombination event responsible for segregation.

pubmed23n0001_137

Involvement of the small intestine in systemic mast cell disease.

A patient is reported with mast cell infiltration of the small intestine in the absence of the skin involvement characteristic of mast cell disease. She also had subtotal villous atrophy responsive to a gluten-free diet. Criteria for diagnosing mast cell disease of the small intestine are proposed. The literature of small intestinal mast cell disease is reviewed and the relationship to coeliac disease is discussed.

Involvement of the small intestine in systemic mast cell disease. A patient is reported with mast cell infiltration of the small intestine in the absence of the skin involvement characteristic of mast cell disease. She also had subtotal villous atrophy responsive to a gluten-free diet. Criteria for diagnosing mast cell disease of the small intestine are proposed. The literature of small intestinal mast cell disease is reviewed and the relationship to coeliac disease is discussed.

pubmed23n0001_138

Gastric pH and microflora of normal and diarrhoeic infants.

The microflora and pH of gastric contents were determined in breast-fed and in bottle-fed normal infants, in well nourished infants with acute diarrhoea and in infants with chronic diarrhoea and protein-calorie malnutrition. The last group of infants was reevaluated after recovery from diarrhoea and protein-calorie malnutrition. A bactericidal pH effect below 2-5 was observed. Bottle-fed controls had low pH values and low bacterial concentrations, whereas infants with chronic diarrhoea and protein-calorie malnutrition had high pH values and bacterial overgrowth, essentially of Gram-negative bacilli. After recovery, the only remaining alteration was the frequent isolation of yeast-like fungi in low concentrations. Infants with acute diarrhoea, except for the isolation more frequently of yeast-like fungi, presented no alterations; this seems to indicate that pH alterations and Gram-negative bacilli overgrowth occurred during the evolution of the disease to a chronic state. Breast-fed normal infants had hydrogen-ion concentrations similar to those of the chronic diarrhoea group, but without Gram-negative bacilli overgrowth, suggesting that other factors, besides pH, regulate bacterial growth in the gastric contents of these groups of infants.

Gastric pH and microflora of normal and diarrhoeic infants. The microflora and pH of gastric contents were determined in breast-fed and in bottle-fed normal infants, in well nourished infants with acute diarrhoea and in infants with chronic diarrhoea and protein-calorie malnutrition. The last group of infants was reevaluated after recovery from diarrhoea and protein-calorie malnutrition. A bactericidal pH effect below 2-5 was observed. Bottle-fed controls had low pH values and low bacterial concentrations, whereas infants with chronic diarrhoea and protein-calorie malnutrition had high pH values and bacterial overgrowth, essentially of Gram-negative bacilli. After recovery, the only remaining alteration was the frequent isolation of yeast-like fungi in low concentrations. Infants with acute diarrhoea, except for the isolation more frequently of yeast-like fungi, presented no alterations; this seems to indicate that pH alterations and Gram-negative bacilli overgrowth occurred during the evolution of the disease to a chronic state. Breast-fed normal infants had hydrogen-ion concentrations similar to those of the chronic diarrhoea group, but without Gram-negative bacilli overgrowth, suggesting that other factors, besides pH, regulate bacterial growth in the gastric contents of these groups of infants.

pubmed23n0001_139

[Biphasic (ulcer-forming and ulcer-preventing) effect of adrenaline in rats].

Adrenaline-induced gastric ulceration was studied in rats. Adrenaline in high doses caused gastric ulcer, which was completely blocked by pretreatment with alpha-blockers (phenoxybenzamine, dibenamine), but not by pretreatment with propranolol or atropine, nor by vagotomy, hypophysectomy or adrenalectomy. After successive administration of adrenaline, once daily for 7 days, however, no gastric ulcer was observed. Recovery from the ulcerogenic action of adrenaline was seen after 4 weeks withdrawal. Pretreatment with a small dose of adrenaline inhibited the ulcerogenic action of a high dose of adrenaline. Pretreatment with reserpine, pyrogallol or iproniazid inhibited the action of adrenaline. It is concluded that adrenaline has a biphasic effect on gastric ulceration, the ulcerogenic action is due to its alpha-action and antiulcerogenic effect is due to development of tachyphylaxis.

[Biphasic (ulcer-forming and ulcer-preventing) effect of adrenaline in rats]. Adrenaline-induced gastric ulceration was studied in rats. Adrenaline in high doses caused gastric ulcer, which was completely blocked by pretreatment with alpha-blockers (phenoxybenzamine, dibenamine), but not by pretreatment with propranolol or atropine, nor by vagotomy, hypophysectomy or adrenalectomy. After successive administration of adrenaline, once daily for 7 days, however, no gastric ulcer was observed. Recovery from the ulcerogenic action of adrenaline was seen after 4 weeks withdrawal. Pretreatment with a small dose of adrenaline inhibited the ulcerogenic action of a high dose of adrenaline. Pretreatment with reserpine, pyrogallol or iproniazid inhibited the action of adrenaline. It is concluded that adrenaline has a biphasic effect on gastric ulceration, the ulcerogenic action is due to its alpha-action and antiulcerogenic effect is due to development of tachyphylaxis.

pubmed23n0001_140

[Comparison of drug effects on the isolated rat colon and duodenum].

Adrenaline and isoproterenol elicited nearly maximal relaxation of the colon even in small doses, whereas increase in the doses caused greater relaxation in the duodenum. In the colon, these drugs prevented, to a great extent the contraction induced by acetylcholine (ACh) and serotonin but in the duodenum were totally ineffective. Dibenamine and propranolol reduced adrenaline- and isoproterenol-induced relaxation in the duodenum, though propranolol decreased the relaxation caused by isoproterenol. Atropine prevented ACh-induced contraction in both the colon and duodenum in the same way. After 2-bromolysergic acid diethylamide, duodenal contraction caused by ACh or serotonin decreased by over 70%; however, the contraction of the colon was not significantly inhibited. Methysergide had similar effects, but to a lesser degree. In calcium-free bathing fluid without addition of Na2EDTA, ACh and prostaglandin E1 elicited contraction in the colon, but not in the duodenum.

[Comparison of drug effects on the isolated rat colon and duodenum]. Adrenaline and isoproterenol elicited nearly maximal relaxation of the colon even in small doses, whereas increase in the doses caused greater relaxation in the duodenum. In the colon, these drugs prevented, to a great extent the contraction induced by acetylcholine (ACh) and serotonin but in the duodenum were totally ineffective. Dibenamine and propranolol reduced adrenaline- and isoproterenol-induced relaxation in the duodenum, though propranolol decreased the relaxation caused by isoproterenol. Atropine prevented ACh-induced contraction in both the colon and duodenum in the same way. After 2-bromolysergic acid diethylamide, duodenal contraction caused by ACh or serotonin decreased by over 70%; however, the contraction of the colon was not significantly inhibited. Methysergide had similar effects, but to a lesser degree. In calcium-free bathing fluid without addition of Na2EDTA, ACh and prostaglandin E1 elicited contraction in the colon, but not in the duodenum.

pubmed23n0001_141

[Monoamine oxidase (XXXVI). Characteristics of benzylamine oxidase in the dog serum].

Enzymic properties of monoamine oxidase (MAO) in dog serum were studied and the following results were obtained. Some of enzymic properties of MAO in dog serum differed from that of mitochondrial MAO. When dog serum was fractionated by ammonium sulfate, proteins were concentrated in two fractions, such as 25 approximately 33% and 67 approximately 80% of saturated ammonium sulfate fraction, while MAO activity was concentrated in 40 approximately 50% of saturated ammonium sulfate fraction. The reaction rate of MAO in dog serum was found to be proportional to enzyme concentration. The optimum pH of MAO in dog serum was 7.0 which differed from that of MAO in rabbit serum (pH 8.0). Tris-HCl buffer strongly inhibited MAO activity in dog serum. When benzylamine was used as substrate, the highest activity was obtained compared with the other substrate used. The activities with butylamine, amylamine, beta-phenylethylamine and tyramine showed about 30% while tryptamine and serotonin showed 3 approximately 10% compared to that with benzymlamine as substrate. The value of pI50 of catron was about 3 X 10(-6) M and that of harmaline was about 3 X 10(-5) M, but pargyline did not inhibit MAO activity in dog serum at the concentration of 1 X 10(-4) M.

[Monoamine oxidase (XXXVI). Characteristics of benzylamine oxidase in the dog serum]. Enzymic properties of monoamine oxidase (MAO) in dog serum were studied and the following results were obtained. Some of enzymic properties of MAO in dog serum differed from that of mitochondrial MAO. When dog serum was fractionated by ammonium sulfate, proteins were concentrated in two fractions, such as 25 approximately 33% and 67 approximately 80% of saturated ammonium sulfate fraction, while MAO activity was concentrated in 40 approximately 50% of saturated ammonium sulfate fraction. The reaction rate of MAO in dog serum was found to be proportional to enzyme concentration. The optimum pH of MAO in dog serum was 7.0 which differed from that of MAO in rabbit serum (pH 8.0). Tris-HCl buffer strongly inhibited MAO activity in dog serum. When benzylamine was used as substrate, the highest activity was obtained compared with the other substrate used. The activities with butylamine, amylamine, beta-phenylethylamine and tyramine showed about 30% while tryptamine and serotonin showed 3 approximately 10% compared to that with benzymlamine as substrate. The value of pI50 of catron was about 3 X 10(-6) M and that of harmaline was about 3 X 10(-5) M, but pargyline did not inhibit MAO activity in dog serum at the concentration of 1 X 10(-4) M.

pubmed23n0001_142

[Combined use of bucolome and pyrazolone derivatives (1). Pharmacological activities and blood concentration].

A combination of two or more drugs may exert a drug-drug interaction, in which case the effect can be potentiated or antagonized. Such synergistic effects are well known in the case of pyrabital (barbital + aminopyrine) or irgapyrine (phenylbutazone + aminopyrine). Bucolome (BCP), a non-steroidal anti-inflammatory agent, has the chemical structure of a barbiturate and also resembles the formula of pheylbutazone. Thus the influence of BCP combination on the pharmacological activities of various pyrazolone derivatives was examined. BCP potentiated the analgesic and antipyretic effects of 4-aminoantipyrine (4A), methylaminoantipyrine (MA), aminopyrine (AM) and isopropylaminoantipyrine (IPA), which were substituted by the alkylamino group at 4-position of the pyrazolone ring. This potentiation occurred when the dose of BCP exceeded that of the pyrazolones, and was especially marked when combination ratio of BCP exceeded that of the pyrazolones, and was especially marked when combination ratio of BCP and pyrazolone was 2:1 mola. The analgesic effects of antipyrine (AN), isopropylantipyrine (IP) and aminopropylone (AP), which were substituted by alkyl group or aminoacylamino group at 4-position, were not potentiated by BCP in any combination ratio. Most pyrazolones showed additive acute toxicity in their combination with BCP, but acute toxicities of 4A and AM, which were potentiated in analgesic effects, were decreased and antagonized when combined with BCP. The plasma concentration of AM was increased and prolonged by BCP, while that of IP remained much the same. These results suggest that the pharmacological activities are associated with certain molecular interactions between BCP and pyrazolones, which are substituted by the alkylamino group at 4-position of the pyrazolone ring.

[Combined use of bucolome and pyrazolone derivatives (1). Pharmacological activities and blood concentration]. A combination of two or more drugs may exert a drug-drug interaction, in which case the effect can be potentiated or antagonized. Such synergistic effects are well known in the case of pyrabital (barbital + aminopyrine) or irgapyrine (phenylbutazone + aminopyrine). Bucolome (BCP), a non-steroidal anti-inflammatory agent, has the chemical structure of a barbiturate and also resembles the formula of pheylbutazone. Thus the influence of BCP combination on the pharmacological activities of various pyrazolone derivatives was examined. BCP potentiated the analgesic and antipyretic effects of 4-aminoantipyrine (4A), methylaminoantipyrine (MA), aminopyrine (AM) and isopropylaminoantipyrine (IPA), which were substituted by the alkylamino group at 4-position of the pyrazolone ring. This potentiation occurred when the dose of BCP exceeded that of the pyrazolones, and was especially marked when combination ratio of BCP exceeded that of the pyrazolones, and was especially marked when combination ratio of BCP and pyrazolone was 2:1 mola. The analgesic effects of antipyrine (AN), isopropylantipyrine (IP) and aminopropylone (AP), which were substituted by alkyl group or aminoacylamino group at 4-position, were not potentiated by BCP in any combination ratio. Most pyrazolones showed additive acute toxicity in their combination with BCP, but acute toxicities of 4A and AM, which were potentiated in analgesic effects, were decreased and antagonized when combined with BCP. The plasma concentration of AM was increased and prolonged by BCP, while that of IP remained much the same. These results suggest that the pharmacological activities are associated with certain molecular interactions between BCP and pyrazolones, which are substituted by the alkylamino group at 4-position of the pyrazolone ring.

pubmed23n0001_143

[Combined use of bucolome and pyrazolone derivatives (II). Complex formation due to interaction between bucolome and pyrazolones].

We have already reported that bucolome (BCP), a non-steroidal anti-inflammatory agent, potentiates significantly the analgesic and antipyretic effects of pyrazolones which are substituted by alkylamino group at 4-position of the pyrazolone ring. Physical and quantum chemistry were applied to the mechanism of this synergistic action. The solubility of BCP was markedly increased in proportion to elevation of aminopyrine (AM) concentration, but not by a combination with isopropylantipyrine (IP). The binding of BCP to bovine serum albumin was slightly inhibited by AM, but not by IP. The mixture of AM and BCP in aqueous media generated optical absorption in the ultraviolet differential spectrum, due to the charge transfer interaction. The results of the infrared or NMR spectrum demonstrated the formation of a hydrogen binding in non-aqueous media between BCP and AM. From the calculation of the charge on an atom, the energy of the highest occupied molecular orbital and the frontier electron density, BCP is considered to be a good electron acceptor. The beta-units of Mho of pyrazolones were found to correlate with the potentiation coefficient of analgesic activity in combination drugs. These results suggest that the complex formation between BCP and pyrazolones is an important factor for the synergism of action and is due to the charge transfer interaction and the hydrogen binding of both molecules.

[Combined use of bucolome and pyrazolone derivatives (II). Complex formation due to interaction between bucolome and pyrazolones]. We have already reported that bucolome (BCP), a non-steroidal anti-inflammatory agent, potentiates significantly the analgesic and antipyretic effects of pyrazolones which are substituted by alkylamino group at 4-position of the pyrazolone ring. Physical and quantum chemistry were applied to the mechanism of this synergistic action. The solubility of BCP was markedly increased in proportion to elevation of aminopyrine (AM) concentration, but not by a combination with isopropylantipyrine (IP). The binding of BCP to bovine serum albumin was slightly inhibited by AM, but not by IP. The mixture of AM and BCP in aqueous media generated optical absorption in the ultraviolet differential spectrum, due to the charge transfer interaction. The results of the infrared or NMR spectrum demonstrated the formation of a hydrogen binding in non-aqueous media between BCP and AM. From the calculation of the charge on an atom, the energy of the highest occupied molecular orbital and the frontier electron density, BCP is considered to be a good electron acceptor. The beta-units of Mho of pyrazolones were found to correlate with the potentiation coefficient of analgesic activity in combination drugs. These results suggest that the complex formation between BCP and pyrazolones is an important factor for the synergism of action and is due to the charge transfer interaction and the hydrogen binding of both molecules.

pubmed23n0001_144

Fructose 1,6-bisphosphate aldolase activity of Rhizobium species.

FDP aldolase was found to be present in the cell-free extracts of Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii, Rhizobium meliloti, Rhizobium lupini, Rhizobium japonicum and Rhizobium species from Arachis hypogaea and Sesbania cannabina. The enzyme in 3 representative species has optimal activity at pH 8.4 in 0.2M veronal buffer. The enzyme activity was completely lost by treatment at 60 degrees C for 15 min. The Km values were in the range from 2.38 to 4.55 X 10(-6)M FDP. Metal chelating agents inhibited enzyme activity, but monovalent or bivalent metal ions failed to stimulate the activity. Bivalent metal ions in general were rather inhibitory.

Fructose 1,6-bisphosphate aldolase activity of Rhizobium species. FDP aldolase was found to be present in the cell-free extracts of Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii, Rhizobium meliloti, Rhizobium lupini, Rhizobium japonicum and Rhizobium species from Arachis hypogaea and Sesbania cannabina. The enzyme in 3 representative species has optimal activity at pH 8.4 in 0.2M veronal buffer. The enzyme activity was completely lost by treatment at 60 degrees C for 15 min. The Km values were in the range from 2.38 to 4.55 X 10(-6)M FDP. Metal chelating agents inhibited enzyme activity, but monovalent or bivalent metal ions failed to stimulate the activity. Bivalent metal ions in general were rather inhibitory.

pubmed23n0001_145

Prevention of cell agglutination and competence in a genetically transformable strain of Pneumococcus by D-glucosamine and D-galactosamine.

In the presence of amino sugars D-glucosamine and D-galactosamine no spontaneous competence could be observed in the highly transformable R6bd strain of Pneumococcus or it was decreased by several orders of magnitude. The highest inhibition of competence was detected when the amino sugar at a concentration 5 mg/ml of the medium was added not only to the transformation but also to the pretransformation medium. After a 150 min growth in the transformation medium in the presence of the amino sugar a 3--4-fold greater number of cells (as a viable count) could be detected as compared with the control without the amino sugar. It was found microscopically that the amino sugar prevents natural agglutination, which normally occurs in the competent culture. The role of specific amino sugar determinants for binding of the competence factor on the cell surface and the resulting inhibitory effect of these sugars on the development of competence are discussed.

Prevention of cell agglutination and competence in a genetically transformable strain of Pneumococcus by D-glucosamine and D-galactosamine. In the presence of amino sugars D-glucosamine and D-galactosamine no spontaneous competence could be observed in the highly transformable R6bd strain of Pneumococcus or it was decreased by several orders of magnitude. The highest inhibition of competence was detected when the amino sugar at a concentration 5 mg/ml of the medium was added not only to the transformation but also to the pretransformation medium. After a 150 min growth in the transformation medium in the presence of the amino sugar a 3--4-fold greater number of cells (as a viable count) could be detected as compared with the control without the amino sugar. It was found microscopically that the amino sugar prevents natural agglutination, which normally occurs in the competent culture. The role of specific amino sugar determinants for binding of the competence factor on the cell surface and the resulting inhibitory effect of these sugars on the development of competence are discussed.

pubmed23n0001_146

Effect of some aldoses on growth of Saccharomyces cerevisiae inhibited with molybdenum.

The inhibitory effect of molybdenum ions on growth of yeasts at pH 5.5 was found to be decreased by aldoses in the following order: D-talose greater than L-mannose greater than L-ribose greater than D-lyxose greater than L-galactose greater than L-arabinose greater than L-glucose greater than L-xylose. Increased concentrations of molybdenum brought about morphological changes of yeast cells. Cells grown under these conditions were smaller, had thicker walls and formed clusters.

Effect of some aldoses on growth of Saccharomyces cerevisiae inhibited with molybdenum. The inhibitory effect of molybdenum ions on growth of yeasts at pH 5.5 was found to be decreased by aldoses in the following order: D-talose greater than L-mannose greater than L-ribose greater than D-lyxose greater than L-galactose greater than L-arabinose greater than L-glucose greater than L-xylose. Increased concentrations of molybdenum brought about morphological changes of yeast cells. Cells grown under these conditions were smaller, had thicker walls and formed clusters.

pubmed23n0001_147

Transport properties of membrane vesicles from Acholeplasma laidlawii. II. Kinetic characteristics and specificity of glucose transport system.

The glucose transport system of membrane vesicles isolated from Acholeplasma laidlawii is saturable, with a Km of 21.2 mum and V of 0.68 nmol min-1 (mg protein)-1. The process is pH-dependent and a break occurs in the Arrhenius plot at 15 degrees C. Exogenous substrates did not stimulate glucose transport probably due to their inability to penetrate into membrane vesicles. 3-O-Methylglucose and 6-deoxyglucose competitively inhibited glucose transport. Maltose inhibited transport of glucose noncompetitively. These sugars also elicited glucose efflux from preloaded membrane vesicles.

Transport properties of membrane vesicles from Acholeplasma laidlawii. II. Kinetic characteristics and specificity of glucose transport system. The glucose transport system of membrane vesicles isolated from Acholeplasma laidlawii is saturable, with a Km of 21.2 mum and V of 0.68 nmol min-1 (mg protein)-1. The process is pH-dependent and a break occurs in the Arrhenius plot at 15 degrees C. Exogenous substrates did not stimulate glucose transport probably due to their inability to penetrate into membrane vesicles. 3-O-Methylglucose and 6-deoxyglucose competitively inhibited glucose transport. Maltose inhibited transport of glucose noncompetitively. These sugars also elicited glucose efflux from preloaded membrane vesicles.

pubmed23n0001_148

Transport of 4-deoxy- and 6-deoxy-D-glucose in baker's yeast.

Tritium-labelled 4-deoxy-D-glucose (4-dglc) and 6-deoxy-D-glucose (6-dgcl) were prepared by catalytic hydrogenolysis of the corresponding deoxyiodo derivatives with gaseous tritium. The two sugars are transported into Saccharomyces cerevisiae by both the constitutive glucose and the inducible galactose carrier. Uranyl ions are powerful inhibitors. The pH optimum in uninduced cells lies at 5.5 for both sugars, the apparent activation energies (between 15 and 35 degrees C) are 25.1 kJ/mol and 16.5 kJ/mol, respectively. The steady-state intracellular concentration of both sugars is less than the extracellular one (no uphill transport). Neither of them is a substrate of yeast hexokinase. 4-Deoxy-D-glucose undergoes a dinitrophenol-sensitive conversion to an unknown metabolite which is not phosphorylated and may represent one of its oxidation products.

Transport of 4-deoxy- and 6-deoxy-D-glucose in baker's yeast. Tritium-labelled 4-deoxy-D-glucose (4-dglc) and 6-deoxy-D-glucose (6-dgcl) were prepared by catalytic hydrogenolysis of the corresponding deoxyiodo derivatives with gaseous tritium. The two sugars are transported into Saccharomyces cerevisiae by both the constitutive glucose and the inducible galactose carrier. Uranyl ions are powerful inhibitors. The pH optimum in uninduced cells lies at 5.5 for both sugars, the apparent activation energies (between 15 and 35 degrees C) are 25.1 kJ/mol and 16.5 kJ/mol, respectively. The steady-state intracellular concentration of both sugars is less than the extracellular one (no uphill transport). Neither of them is a substrate of yeast hexokinase. 4-Deoxy-D-glucose undergoes a dinitrophenol-sensitive conversion to an unknown metabolite which is not phosphorylated and may represent one of its oxidation products.

pubmed23n0001_149

Gluconic acid production by Penicillium puberulum.

Twenty-five Penicillium species isolated from Egyptian soil were examined for their ability to produce gluconic acid in surface culture. Of the eight species capable of producing gluconic acid, Penicillium puberulum gave the maximum yield (91% gluconic acid from glucose after 7 days of fermentation with 3% CaCO3). Peptone was the best nitrogen source for acid fermentation and glucose was superior to sucrose. Addition of low concentrations of KH2PO4 and MgSO4 - 7 H2O stimulated acid production. An initial pH of 6.1 was most favourable for acid accumulation and addition of CaCO3 was necessary for maximum acid production.

Gluconic acid production by Penicillium puberulum. Twenty-five Penicillium species isolated from Egyptian soil were examined for their ability to produce gluconic acid in surface culture. Of the eight species capable of producing gluconic acid, Penicillium puberulum gave the maximum yield (91% gluconic acid from glucose after 7 days of fermentation with 3% CaCO3). Peptone was the best nitrogen source for acid fermentation and glucose was superior to sucrose. Addition of low concentrations of KH2PO4 and MgSO4 - 7 H2O stimulated acid production. An initial pH of 6.1 was most favourable for acid accumulation and addition of CaCO3 was necessary for maximum acid production.

pubmed23n0001_150

A simple method of isolation and purification of cultures of wood-rotting fungi.

A simple method of isolation and purification of cultures of higher fungi, mainly wood-rotting fungi, without special requirements for the presence of nitrogencontaining nutrients is described. Parts of fruiting bodies, spores or infected wood is inoculated on Petri dishes with an agar medium of the following composition: 1.0 g KH2PO4, 0.2 g MgSO4 - 7 H2O, 0.1 g caSO4, 0.01 g Fe2(SO4)3, 10.0 g glucose, tap water to 1 litre; 20.0 g agar. This medium does not suit most of the contaminants but fungal hyphae overgrow the whole surface of the dish so that a purified culture can be obtained from parts distant from the inoculation site.

A simple method of isolation and purification of cultures of wood-rotting fungi. A simple method of isolation and purification of cultures of higher fungi, mainly wood-rotting fungi, without special requirements for the presence of nitrogencontaining nutrients is described. Parts of fruiting bodies, spores or infected wood is inoculated on Petri dishes with an agar medium of the following composition: 1.0 g KH2PO4, 0.2 g MgSO4 - 7 H2O, 0.1 g caSO4, 0.01 g Fe2(SO4)3, 10.0 g glucose, tap water to 1 litre; 20.0 g agar. This medium does not suit most of the contaminants but fungal hyphae overgrow the whole surface of the dish so that a purified culture can be obtained from parts distant from the inoculation site.

pubmed23n0001_151

[Studies on low volume priming heart lung bypass (author's transl)].

This report concerns the feasibility of low volume priming extracorporeal circulation. Through this study, the bubble oxygenator with Zuhdi's heat exchange was used. Moderate hypothermia with surface cooling and hemodilution perfusion with 5 per cent D/W was evaluated in 32 mongrel dogs and 16 clinical open heart cases. The results obtained here were as follow: 1) Body temperature reduction by surface cooling before bypass provided more even cooling than did core cooling by low flow partial bypass alone. 2) In regard to cardiac loading on returning the whole perfusate of the circuit to patient, approximately 20 ml/kg of 5 per cent D/W was feasible as a priming solution. 3) To reduce the blood visicosity, hemodilution technique with 5 per cent D/W was superior, and hemodilution effect during postoperative periods was temporaly. 4) The excess lactate volume postulated by Huckabee was a available index to evaluate metabolic acidosis during the extracorporeal circulation. 5) With aid of surface cooling, the acid-base balance during perfusion was kept to lesser extent than that of core cooling only. 6) This study indicated that the low priming perfusion in conjunction with surface cooling hypothermia was a reliable technique for the open heart operation and may be applied in more prolonged perfusion.

[Studies on low volume priming heart lung bypass (author's transl)]. This report concerns the feasibility of low volume priming extracorporeal circulation. Through this study, the bubble oxygenator with Zuhdi's heat exchange was used. Moderate hypothermia with surface cooling and hemodilution perfusion with 5 per cent D/W was evaluated in 32 mongrel dogs and 16 clinical open heart cases. The results obtained here were as follow: 1) Body temperature reduction by surface cooling before bypass provided more even cooling than did core cooling by low flow partial bypass alone. 2) In regard to cardiac loading on returning the whole perfusate of the circuit to patient, approximately 20 ml/kg of 5 per cent D/W was feasible as a priming solution. 3) To reduce the blood visicosity, hemodilution technique with 5 per cent D/W was superior, and hemodilution effect during postoperative periods was temporaly. 4) The excess lactate volume postulated by Huckabee was a available index to evaluate metabolic acidosis during the extracorporeal circulation. 5) With aid of surface cooling, the acid-base balance during perfusion was kept to lesser extent than that of core cooling only. 6) This study indicated that the low priming perfusion in conjunction with surface cooling hypothermia was a reliable technique for the open heart operation and may be applied in more prolonged perfusion.

pubmed23n0001_152

[Studies on extracorporeal circulation with large volume hemodilution using lactate ringer's solution and low molecular weight dextran: alterations of acid-base balance associated with intentional hemodilution (author's transl)].

Twenty mongrel dogs, weighing between 7.5 and 13.0 kg were used to investigate the percentage limits permissible for hemodilution using a double-helical reservoir heart-lung machine which has a 1,100 ml of priming volume. In both 40 and 50 per cent groups of intentional hemodilution by 30 minute extracorporeal circulation, remarkable anemia was inevitable and recovery was extremely slow, especially in the 50 per cent dilution group. In both 40 and 50 per cent groups of intentional hemodilutions by 30 minute extracorporeal circulation, metabolic acidosis was observed. In 50 per cent group of intentional hemodilution, no improvement of metabolic acidosis was observed even after perfusion. When sodium bicarbonate was administered to 40 per cent hemodilution group, minimum alterations of acid-base balance and of serum electrolytes were observed during and after extracorporeal perfusion. When sodium bicarbonate was administered to 50 per cent hemodilution group, metabolic acidosis was more evident than in 40 per cent hemodilution group accompanied with an increase in serum sodium concentration and a decrease in serum chloride concentration. These data qualify the use of 40 per cent intentional hemodilution using Lactate Ringer's solution or low molecular weight dextran for 30 minute extracorporeal circulation when sodium bicarbonate is administered in adequate amounts.

[Studies on extracorporeal circulation with large volume hemodilution using lactate ringer's solution and low molecular weight dextran: alterations of acid-base balance associated with intentional hemodilution (author's transl)]. Twenty mongrel dogs, weighing between 7.5 and 13.0 kg were used to investigate the percentage limits permissible for hemodilution using a double-helical reservoir heart-lung machine which has a 1,100 ml of priming volume. In both 40 and 50 per cent groups of intentional hemodilution by 30 minute extracorporeal circulation, remarkable anemia was inevitable and recovery was extremely slow, especially in the 50 per cent dilution group. In both 40 and 50 per cent groups of intentional hemodilutions by 30 minute extracorporeal circulation, metabolic acidosis was observed. In 50 per cent group of intentional hemodilution, no improvement of metabolic acidosis was observed even after perfusion. When sodium bicarbonate was administered to 40 per cent hemodilution group, minimum alterations of acid-base balance and of serum electrolytes were observed during and after extracorporeal perfusion. When sodium bicarbonate was administered to 50 per cent hemodilution group, metabolic acidosis was more evident than in 40 per cent hemodilution group accompanied with an increase in serum sodium concentration and a decrease in serum chloride concentration. These data qualify the use of 40 per cent intentional hemodilution using Lactate Ringer's solution or low molecular weight dextran for 30 minute extracorporeal circulation when sodium bicarbonate is administered in adequate amounts.

pubmed23n0001_153

Purification and characterization of phosphodiesterase from Crotalus venom.

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.

Purification and characterization of phosphodiesterase from Crotalus venom. A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.

pubmed23n0001_154

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.

Limited hydrolysis of tRNA by phosphodiesterase. Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.

pubmed23n0001_155

Ecdysone Oxidase, an enzyme from the blowfly Calliphora erythrocephala (Meigen).

In the blowfly, the formation of 3-dehydroecdysone from the insect molting hormone ecdysone is catalyzed by an enzyme which carries hydrogen from ecdysone and ecdysterone to oxygen. The enzyme is therefore called "ecdysone oxidase". Two methods are described for the detection of ecdysone oxidase activity, one using a radiolabelled substrate which is separated from the product by thin-layer chromatography after the reaction, and the other using dichloroindophenol, which is discoloured by the redox reaction. The ecdysone oxidase is purified by a factor of 2200 from prepupae of Calliphora erythrocephala using salt precipitation and ion exchange chromatography. The ecdysone oxidase has a Km value for ecdysone of 42muM. The pH optimum is 6.5. The temperature optimum lies at 45 degrees C. The ecdysone oxidase has a molecular weight of 240000.

Ecdysone Oxidase, an enzyme from the blowfly Calliphora erythrocephala (Meigen). In the blowfly, the formation of 3-dehydroecdysone from the insect molting hormone ecdysone is catalyzed by an enzyme which carries hydrogen from ecdysone and ecdysterone to oxygen. The enzyme is therefore called "ecdysone oxidase". Two methods are described for the detection of ecdysone oxidase activity, one using a radiolabelled substrate which is separated from the product by thin-layer chromatography after the reaction, and the other using dichloroindophenol, which is discoloured by the redox reaction. The ecdysone oxidase is purified by a factor of 2200 from prepupae of Calliphora erythrocephala using salt precipitation and ion exchange chromatography. The ecdysone oxidase has a Km value for ecdysone of 42muM. The pH optimum is 6.5. The temperature optimum lies at 45 degrees C. The ecdysone oxidase has a molecular weight of 240000.

pubmed23n0001_156

Using a hospital for desensitization of an outpatient's illness-related fears.

An outpatient with phobias related to enclosed places, hospitals, doctors, and cancer was treated by systematic desensitization; the facilities of a general hospital were used for part of the process. Steps in treatment included securing a complete psychiatric and social history, teaching the patient relaxation therapy techniques, and establishing a hierarchy of anxiety-provoking stimuli specifically related to the patient's fears. After desensitization the patient was able to enter the hospital for tests for a physical ailment and showed a general decrease in her fears.

Using a hospital for desensitization of an outpatient's illness-related fears. An outpatient with phobias related to enclosed places, hospitals, doctors, and cancer was treated by systematic desensitization; the facilities of a general hospital were used for part of the process. Steps in treatment included securing a complete psychiatric and social history, teaching the patient relaxation therapy techniques, and establishing a hierarchy of anxiety-provoking stimuli specifically related to the patient's fears. After desensitization the patient was able to enter the hospital for tests for a physical ailment and showed a general decrease in her fears.

pubmed23n0001_157

Analysis of immunosuppression generated by the graft-versus-host reaction. I. A suppressor T-cell component studied in vivo.

The kinetics and cellular characteristics of immunosuppression in (CBA-p X C57/Bl)F1 mice during graft-versus-host (GVH) reaction induced with spleen cells from either parental strain have been investigated. Effects on PFC responses to thymus-dependent (chicken red blood cells) and independent (levan) antigens have been compared. The unresponsive state which developed in GVH mice was expressed to comparable extent by their spleen cells following transfer to irradiated recipients. Furthermore, GVH spleen cells suppressed normal spleen cells in a mixed transfer system, but this effect was lost completely by day 21 whereas the original donors (or their cells) were still totally refractory after 80 days. This active suppressor effect was found to be mediated by T cells of perental origin based on cell fractionation analysis and selective deletion of donor or host cells by alloimmune attack. The delayed transfer of GVH cells to irradiated repopulated recipients challenged with antigen, indicated that suppressor T cells exert an anti-mitotic influence on antigen-stimulated B-cell proliferation. Supplementation of macrophage-depleted, anti-theta-treated GVH spleen cells with purified normal T cells demonstrated that B cells in GVH mice are normally reactive even when active suppression by T cells is no longer demonstrable. The likelihood that this later phase of immunosuppression is attributable to another mechanism is discussed.

Analysis of immunosuppression generated by the graft-versus-host reaction. I. A suppressor T-cell component studied in vivo. The kinetics and cellular characteristics of immunosuppression in (CBA-p X C57/Bl)F1 mice during graft-versus-host (GVH) reaction induced with spleen cells from either parental strain have been investigated. Effects on PFC responses to thymus-dependent (chicken red blood cells) and independent (levan) antigens have been compared. The unresponsive state which developed in GVH mice was expressed to comparable extent by their spleen cells following transfer to irradiated recipients. Furthermore, GVH spleen cells suppressed normal spleen cells in a mixed transfer system, but this effect was lost completely by day 21 whereas the original donors (or their cells) were still totally refractory after 80 days. This active suppressor effect was found to be mediated by T cells of perental origin based on cell fractionation analysis and selective deletion of donor or host cells by alloimmune attack. The delayed transfer of GVH cells to irradiated repopulated recipients challenged with antigen, indicated that suppressor T cells exert an anti-mitotic influence on antigen-stimulated B-cell proliferation. Supplementation of macrophage-depleted, anti-theta-treated GVH spleen cells with purified normal T cells demonstrated that B cells in GVH mice are normally reactive even when active suppression by T cells is no longer demonstrable. The likelihood that this later phase of immunosuppression is attributable to another mechanism is discussed.

pubmed23n0001_158

Pneumococcal type-associated variability in alternate complement pathway activation.

Opsonization of Streptococcus pneumoniae may be mediated by the alternate complement pathway. To study the importance of this interaction to human disease, complement consumption by pneumococci of various serotypes was measured in humwn serum chelated with ethyleneglycoltraacetic acid, a substance that blocks the classic but not the alternate complement pathwway. Serotype I, in contrast to all other types studied, lacked ability to consume complement in this system. The ability for serotypes III, IV, and VIII to activate the alternate pathway could be eliminated by prior serum absorption at O C with they type in question, a condition that would remove antibody but not complement. Types VII, XII, XIV, and XXV readily activated the alternate pathway in unabsorbed and absorbed sera. Differences could not be related to properties of the capsules. It was concluded that types I, III, IV, and VIII lack intrinsic ability to activate and thus be opsonized by the alternate complement pathway, although types III, IV, and VIII can do so in concert with specific antibody. The fact that these same types are especially prominent in human disease suggests that the ability to evade opsonization by the alternate complement pathway in pre-antibody phases of infection may be a virulence factor in pneumococci.

Pneumococcal type-associated variability in alternate complement pathway activation. Opsonization of Streptococcus pneumoniae may be mediated by the alternate complement pathway. To study the importance of this interaction to human disease, complement consumption by pneumococci of various serotypes was measured in humwn serum chelated with ethyleneglycoltraacetic acid, a substance that blocks the classic but not the alternate complement pathwway. Serotype I, in contrast to all other types studied, lacked ability to consume complement in this system. The ability for serotypes III, IV, and VIII to activate the alternate pathway could be eliminated by prior serum absorption at O C with they type in question, a condition that would remove antibody but not complement. Types VII, XII, XIV, and XXV readily activated the alternate pathway in unabsorbed and absorbed sera. Differences could not be related to properties of the capsules. It was concluded that types I, III, IV, and VIII lack intrinsic ability to activate and thus be opsonized by the alternate complement pathway, although types III, IV, and VIII can do so in concert with specific antibody. The fact that these same types are especially prominent in human disease suggests that the ability to evade opsonization by the alternate complement pathway in pre-antibody phases of infection may be a virulence factor in pneumococci.

pubmed23n0001_159

Conditions for production, and some characteristics, of mycobacterial growth inhibitory factor produced by spleen cells from mice immunized with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis.

Mycobacterial growth inhibitory factor (MycoIF), found in supernatant fluids of mouse spleen cell cultures that have been stimulated in vitro with homologous antigen, inhibited the intracellular multiplication of virulent tubercle bacilli within normal mouse peritoneal macrophages in vitro. Antigenically stimulated H37Ra-immunized mouse spleen cells required 72 h of incubation to produce supernatant fluids that would cause intracellular inhibition. Supernatant fluids from 48-h mouse spleen cell cultures were not able to produce intracellular inhibition. Investigation of the culture conditions showed that at lease 1.0% human serum was required in the tissue culture medium for the production of MycoIF by spleen cells from immunized mice. MycoIF activity was noted only in supernatant fluids from spleen cell cultures incubated with antigen for 72 h. MycoIF was nondialyzable and unaffected by freezing, lyophilization, or incubation at 60 C for 30 min. However, MycoIF was inactivated after incubation at 80 C for 30 min. MycoIF was unaffected by low hydrogen ion concentrations (pH 7 to 12), but exposure to higher hydrogen ion concentrations (pH 6, pH 5) significantly decrease MycoIF activity, and exposure to pH 4 to 2 abolished all activity. Supernatant fluids diluted 1:32 were still able to produce significant intracellular inhibition of growth of virulent tubercle bacilli.

Conditions for production, and some characteristics, of mycobacterial growth inhibitory factor produced by spleen cells from mice immunized with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis. Mycobacterial growth inhibitory factor (MycoIF), found in supernatant fluids of mouse spleen cell cultures that have been stimulated in vitro with homologous antigen, inhibited the intracellular multiplication of virulent tubercle bacilli within normal mouse peritoneal macrophages in vitro. Antigenically stimulated H37Ra-immunized mouse spleen cells required 72 h of incubation to produce supernatant fluids that would cause intracellular inhibition. Supernatant fluids from 48-h mouse spleen cell cultures were not able to produce intracellular inhibition. Investigation of the culture conditions showed that at lease 1.0% human serum was required in the tissue culture medium for the production of MycoIF by spleen cells from immunized mice. MycoIF activity was noted only in supernatant fluids from spleen cell cultures incubated with antigen for 72 h. MycoIF was nondialyzable and unaffected by freezing, lyophilization, or incubation at 60 C for 30 min. However, MycoIF was inactivated after incubation at 80 C for 30 min. MycoIF was unaffected by low hydrogen ion concentrations (pH 7 to 12), but exposure to higher hydrogen ion concentrations (pH 6, pH 5) significantly decrease MycoIF activity, and exposure to pH 4 to 2 abolished all activity. Supernatant fluids diluted 1:32 were still able to produce significant intracellular inhibition of growth of virulent tubercle bacilli.

pubmed23n0001_160

Effect of pneumococci on blood clotting, platelets, and polymorphonuclear leukocytes.

Infections due to Streptococcus pneumoniae and products from the organism have been associated with alterations in blood clotting and function of platelets. Pneumococci and pneumococcal polysaccharide shortened the clotting times of whole blood, platelet-rich plasma (PRP), and platelet-poor plasma (PPP) in vitro. Clotting times of PPP and PRP from C6-deficient animals were likewise decreased. The bacteria had no effect on the one-stage prothrombin time or the partial thromboplastin time when the organisms were used as activating agents. Platelets aggregated in the presence of pneumococci, but aggregation was prevented by the addition of cyclic adenosine 3', 5'-monophosphate (cAMP). Furthermore, cAMP corrected the shortened clotting time of PRP in the presence of pneumococci. The clumping and release of polymorphonuclear coagulant that was induced by pneumococci was not prevented by cAMP. Thus, pneumococci exert several dose-dependent thromboplastic effects: (i) release of platelet thromboplastic substances; (ii) a direct thromboplastic effect; and (iii) release of polymorphonuclear coagulant.

Effect of pneumococci on blood clotting, platelets, and polymorphonuclear leukocytes. Infections due to Streptococcus pneumoniae and products from the organism have been associated with alterations in blood clotting and function of platelets. Pneumococci and pneumococcal polysaccharide shortened the clotting times of whole blood, platelet-rich plasma (PRP), and platelet-poor plasma (PPP) in vitro. Clotting times of PPP and PRP from C6-deficient animals were likewise decreased. The bacteria had no effect on the one-stage prothrombin time or the partial thromboplastin time when the organisms were used as activating agents. Platelets aggregated in the presence of pneumococci, but aggregation was prevented by the addition of cyclic adenosine 3', 5'-monophosphate (cAMP). Furthermore, cAMP corrected the shortened clotting time of PRP in the presence of pneumococci. The clumping and release of polymorphonuclear coagulant that was induced by pneumococci was not prevented by cAMP. Thus, pneumococci exert several dose-dependent thromboplastic effects: (i) release of platelet thromboplastic substances; (ii) a direct thromboplastic effect; and (iii) release of polymorphonuclear coagulant.

pubmed23n0001_161

Some properties of a D-alanine carboxypeptidase in envelope fractions of Neisseria gonorrhoeae.

Envelope preparations of Neisseria gonorrhoeae strain GC1 (a stable, piliated strain of intermediate colony morphology) and type T1 possess a D-alanine carboxypeptidase which releases the terminal alanine residue from the uridine 5'-diphosphate-N-acetyl muramylpentapeptide substrate (isolated from Bacillus cereus T). The D-alanine carboxypeptidase of the GC1 envelopes has a broad pH optimum between pH 8.0 to 10.0. When the molarity of the tris(hydroxymethyl)aminomethane buffer was varied, the activity showed an optimum over the range 0.2 to 0.4 M. Activity was higher (135% of control level) when 20 to 80 mM Mg2+ was present. The Km for the enzyme was 0.25 mM. The D-alanine carboxypeptidase was inhibited by several beta-lactam antibiotics and the 50% inhibitory levels were 10(-8) M penicillin G, 10(-8) M ampicillin, 10(-5) M cloxacillin, and 5 x 10(-7) M methicillin.

Some properties of a D-alanine carboxypeptidase in envelope fractions of Neisseria gonorrhoeae. Envelope preparations of Neisseria gonorrhoeae strain GC1 (a stable, piliated strain of intermediate colony morphology) and type T1 possess a D-alanine carboxypeptidase which releases the terminal alanine residue from the uridine 5'-diphosphate-N-acetyl muramylpentapeptide substrate (isolated from Bacillus cereus T). The D-alanine carboxypeptidase of the GC1 envelopes has a broad pH optimum between pH 8.0 to 10.0. When the molarity of the tris(hydroxymethyl)aminomethane buffer was varied, the activity showed an optimum over the range 0.2 to 0.4 M. Activity was higher (135% of control level) when 20 to 80 mM Mg2+ was present. The Km for the enzyme was 0.25 mM. The D-alanine carboxypeptidase was inhibited by several beta-lactam antibiotics and the 50% inhibitory levels were 10(-8) M penicillin G, 10(-8) M ampicillin, 10(-5) M cloxacillin, and 5 x 10(-7) M methicillin.

pubmed23n0001_162

Host defense against the pneumococcus in T-lymphocyte-deficient, nude mice.

Resistance to pneumococcal infection was tested in T-lymphocyte-deficient, nude (nu/nu) mice. Pneumococcal serum opsonizing activity, in vivo phagocytosis of the pneumococcus, and the mean lethal dose for the pneumococcus were all found to be the same in nude mice as in control (+/+) mice. T-lymphocytes do not appear to play a significant role in the host's defense against the pneumococcus.

Host defense against the pneumococcus in T-lymphocyte-deficient, nude mice. Resistance to pneumococcal infection was tested in T-lymphocyte-deficient, nude (nu/nu) mice. Pneumococcal serum opsonizing activity, in vivo phagocytosis of the pneumococcus, and the mean lethal dose for the pneumococcus were all found to be the same in nude mice as in control (+/+) mice. T-lymphocytes do not appear to play a significant role in the host's defense against the pneumococcus.

pubmed23n0001_163

Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes.

Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (beta-hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C-treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot-cold hemolysis. The addition of ethylenediaminetetraacetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator-induced hemolysis and to hot-cold hemolysis simultaneously, and the degree of lysis of both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot-cold hemolysis were equally insusceptible to chelator-induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator-induced lysis was dependent on the preincubation period with beta-hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator-induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator-induced lysis. The data suggest that exogenously added and/or membrane-bound divalent cations are important for the stability of sphingomyelin-depleted membranes. The phenomenon of hot-cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization.

Phenomenon of hot-cold hemolysis: chelator-induced lysis of sphingomyelinase-treated erythrocytes. Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (beta-hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C-treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot-cold hemolysis. The addition of ethylenediaminetetraacetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator-induced hemolysis and to hot-cold hemolysis simultaneously, and the degree of lysis of both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot-cold hemolysis were equally insusceptible to chelator-induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator-induced lysis was dependent on the preincubation period with beta-hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator-induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator-induced lysis. The data suggest that exogenously added and/or membrane-bound divalent cations are important for the stability of sphingomyelin-depleted membranes. The phenomenon of hot-cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization.

pubmed23n0001_164

In vitro antagonism of the mediators of allergy by a benzopyrano-benzopyran carboxylic acid PR-D-92-EA.

PR-D-92-EA was tested on isolated guinea pig ileum and rat stomach strips for activity against mediators probably released after allergen antibody union. It antagonized the response produced by histamine, bradykinin, serotonin, prostaglandin E2, prostaglandin F2ALPHA and slow-reacting substance of anaphylaxis (SRS-A). The concentrations which blocked 50% of the response were 150, 145, 92, 70, 47, and 32 mug/ml, respectively. This compound may be useful in the treatment of allergic conditions.

In vitro antagonism of the mediators of allergy by a benzopyrano-benzopyran carboxylic acid PR-D-92-EA. PR-D-92-EA was tested on isolated guinea pig ileum and rat stomach strips for activity against mediators probably released after allergen antibody union. It antagonized the response produced by histamine, bradykinin, serotonin, prostaglandin E2, prostaglandin F2ALPHA and slow-reacting substance of anaphylaxis (SRS-A). The concentrations which blocked 50% of the response were 150, 145, 92, 70, 47, and 32 mug/ml, respectively. This compound may be useful in the treatment of allergic conditions.

pubmed23n0001_165

Tyrosine aminotransferase induction in normal and tumor-bearing chickens.

Tyrosine aminotransferase (TAT) induction by glucagon and dexamethasone in the liver of tumor-bearing chickens was studied and compared with induction in healthy animals. The transplantable tumor was caused by inoculation of cells from a cell line induced by MC29 avian leukosis virus. TAT was hardly detectable in tumor tissue of control and dexamethasone-treated chickens, but it was induced by glucagon to levels which were significant although very low when compared to those in host liver or the liver of non-tumor-bearing controls after glucagon treatment. Dexamethasone failed to induce TAT in host liver at 8 A.M. while it significantly indiced TAT in the normal liver at the same time of the day. Similar failure of TAT induction was not detectable when glucagon was used instead of dexamethasone. Furthermore, it was found that diurnal variations in basal and dexamethasone or glucagon-induced TAT levels are considerably mitigated in host liver as compared to those observed in the liver of healthy animals. The possible reasons for these findings are discussed.

Tyrosine aminotransferase induction in normal and tumor-bearing chickens. Tyrosine aminotransferase (TAT) induction by glucagon and dexamethasone in the liver of tumor-bearing chickens was studied and compared with induction in healthy animals. The transplantable tumor was caused by inoculation of cells from a cell line induced by MC29 avian leukosis virus. TAT was hardly detectable in tumor tissue of control and dexamethasone-treated chickens, but it was induced by glucagon to levels which were significant although very low when compared to those in host liver or the liver of non-tumor-bearing controls after glucagon treatment. Dexamethasone failed to induce TAT in host liver at 8 A.M. while it significantly indiced TAT in the normal liver at the same time of the day. Similar failure of TAT induction was not detectable when glucagon was used instead of dexamethasone. Furthermore, it was found that diurnal variations in basal and dexamethasone or glucagon-induced TAT levels are considerably mitigated in host liver as compared to those observed in the liver of healthy animals. The possible reasons for these findings are discussed.

pubmed23n0001_166

A behavior modification training program for staff working with drug addicts.

This paper described a Behavior Modification Training Program, emphasizing self-control, for staff working with drug addicts. The program, which is primarily geared toward the training of paraprofessionals, takes place in ten 1-1/2 hour sessions and includes an overview of behavior modification as well as instruction in techniques of relaxation, desensitization, self-image improvement, behavior analysis, behavior control, assertive training, rational thinking, and how to set up and run similar behavior modification training programs for staff and patients. Since this training began at the New Jersey Neuropsychiatric Institute in November 1971, a total of 898 staff members, mostly paraprofessionals working with addicts, alcoholics, mentally ill patients, and inmates, including 53 from our own institution, 576 persons from other facilities in New Jersey, and 269 from facilities in other states, have been trained, while 2,021 patients have been trained in similar programs. Most of this training has been accomplished by paraprofessionals. Preliminary evaluation data have been promising and the response of participants enthusiastic.

A behavior modification training program for staff working with drug addicts. This paper described a Behavior Modification Training Program, emphasizing self-control, for staff working with drug addicts. The program, which is primarily geared toward the training of paraprofessionals, takes place in ten 1-1/2 hour sessions and includes an overview of behavior modification as well as instruction in techniques of relaxation, desensitization, self-image improvement, behavior analysis, behavior control, assertive training, rational thinking, and how to set up and run similar behavior modification training programs for staff and patients. Since this training began at the New Jersey Neuropsychiatric Institute in November 1971, a total of 898 staff members, mostly paraprofessionals working with addicts, alcoholics, mentally ill patients, and inmates, including 53 from our own institution, 576 persons from other facilities in New Jersey, and 269 from facilities in other states, have been trained, while 2,021 patients have been trained in similar programs. Most of this training has been accomplished by paraprofessionals. Preliminary evaluation data have been promising and the response of participants enthusiastic.

pubmed23n0001_167

A kinetic study of the hydrolysis of N-acetyl dehydroalanine methyl ester.

Hydrolysis rates of N-acetyl dehydroalanine methyl ester (methyl 2-acetamidoacrylate) and related model compounds were measured in aqueous, organic and mixed aqueous media. Adding dimethylsulfoxide (DMSO) to water, retarded hydrolysis of the ester by a factor of 2 to 500, depending on the pH of the medium and concentration of DMSO. Ethanol also slowed hydrolysis, but the effect was not so pronounced. Related studies show that the acetamido group C-N bond of sodium 2-acetamido-acrylate is hydrolyzed only about 1/130 as fast as the ester group C-O bond. Aqueous dimethyl sulfoxide should by a useful medium for synthesis of peptide, amino acid and protein derivatives of N-acetyl dehydroalanine methyl ester.

A kinetic study of the hydrolysis of N-acetyl dehydroalanine methyl ester. Hydrolysis rates of N-acetyl dehydroalanine methyl ester (methyl 2-acetamidoacrylate) and related model compounds were measured in aqueous, organic and mixed aqueous media. Adding dimethylsulfoxide (DMSO) to water, retarded hydrolysis of the ester by a factor of 2 to 500, depending on the pH of the medium and concentration of DMSO. Ethanol also slowed hydrolysis, but the effect was not so pronounced. Related studies show that the acetamido group C-N bond of sodium 2-acetamido-acrylate is hydrolyzed only about 1/130 as fast as the ester group C-O bond. Aqueous dimethyl sulfoxide should by a useful medium for synthesis of peptide, amino acid and protein derivatives of N-acetyl dehydroalanine methyl ester.

pubmed23n0001_168

The ophthalmologist's office: planning and practice. Patient traffic flow and use of paramedical personnel.

In order for the ophthalmologist to achieve the greatest earning power and personal satisfaction, he or she must devise traffic flow techniques that efficiently allow for maximum utilization of all examining areas and paramedical personnel in the office without dehumanizing patients. By delegating as much responsibility as possible to staff, the ophthalmologist will have the opportunity of performing his or her specialized skills to the fullest extent, and therefore will achieve optimal personal gratification. This emotional reward indemnifies the future of private practice, because it can exist only in the presence of a close patient-physician relationship, which is the cornerstone of the private practice of ophthalmology.

The ophthalmologist's office: planning and practice. Patient traffic flow and use of paramedical personnel. In order for the ophthalmologist to achieve the greatest earning power and personal satisfaction, he or she must devise traffic flow techniques that efficiently allow for maximum utilization of all examining areas and paramedical personnel in the office without dehumanizing patients. By delegating as much responsibility as possible to staff, the ophthalmologist will have the opportunity of performing his or her specialized skills to the fullest extent, and therefore will achieve optimal personal gratification. This emotional reward indemnifies the future of private practice, because it can exist only in the presence of a close patient-physician relationship, which is the cornerstone of the private practice of ophthalmology.

pubmed23n0001_169

Computer evaluation of the effect of urecholine on the spontaneous activity of smooth muscle from the urinary bladder of the rabbit.

The spontaneous activity exhibited in vitro by smooth muscle strips from the urinary bladder of the rabbit under the influence of Urecholine in concentrations from 0.3 to 30 muM has been characterized in terms of three parameters: mean tension, frequency of contraction, and contractile deviation, all obtained by computer an alysis. While mean tension and frequency monotonically increased with concentration, the contractile deviation rose to a peak in the vicinity of 3.0 to 6.0 muM and thereafter decreased. At concentrations less than 10 muM the contractile patterns were almost periodic, while at 10 and 30 muM, the patterns became highly erratic. Histologic examination of a limited number of tissues showed longitudinally oriented muscle bundles with multiple anastomosing.

Computer evaluation of the effect of urecholine on the spontaneous activity of smooth muscle from the urinary bladder of the rabbit. The spontaneous activity exhibited in vitro by smooth muscle strips from the urinary bladder of the rabbit under the influence of Urecholine in concentrations from 0.3 to 30 muM has been characterized in terms of three parameters: mean tension, frequency of contraction, and contractile deviation, all obtained by computer an alysis. While mean tension and frequency monotonically increased with concentration, the contractile deviation rose to a peak in the vicinity of 3.0 to 6.0 muM and thereafter decreased. At concentrations less than 10 muM the contractile patterns were almost periodic, while at 10 and 30 muM, the patterns became highly erratic. Histologic examination of a limited number of tissues showed longitudinally oriented muscle bundles with multiple anastomosing.

pubmed23n0001_170

Characteristics of the non-occluded form of a nuclear polyhedrosis virus.

Non-occluded virions of a nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, found in the medium of cell cultures of infected fall armyworm, Spodopter frugiperda, and in the hemolymph of infected S. frugiperda larvae were partially characterized by biological, chemical and physical methods. Also, the rate of appearance of the virions was studied in cell culture and the host insect to determine maximum virion production. Virions obtained from both sources were heat-sensitive, acid-labile and inactivated by several organic solvents. The non-occluded virions found in the insect cell culture fluid and in the hemolymph were identical, and both were enveloped nucleocapsids. Visualization of the fragilely enveloped nucleocapsid was accomplished only after fixation with glutaraldehyde. Differences between the non-occluded and occluded virions of nuclear polyhedrosis viruses are discussed.

Characteristics of the non-occluded form of a nuclear polyhedrosis virus. Non-occluded virions of a nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, found in the medium of cell cultures of infected fall armyworm, Spodopter frugiperda, and in the hemolymph of infected S. frugiperda larvae were partially characterized by biological, chemical and physical methods. Also, the rate of appearance of the virions was studied in cell culture and the host insect to determine maximum virion production. Virions obtained from both sources were heat-sensitive, acid-labile and inactivated by several organic solvents. The non-occluded virions found in the insect cell culture fluid and in the hemolymph were identical, and both were enveloped nucleocapsids. Visualization of the fragilely enveloped nucleocapsid was accomplished only after fixation with glutaraldehyde. Differences between the non-occluded and occluded virions of nuclear polyhedrosis viruses are discussed.

pubmed23n0001_171

Degradation of myxovirus virion RNA by periodate.

Extensively degraded RNA was isolated from virions of influenza virus which had been oxidized with sodium m-periodate. Similarly, although to a lesser extent, RNA isolated from periodate-treated ribonucleoprotein of influenza virus was also degraded. In contrast, influenza virus RNA, if first freed from other virion components, was not degraded by periodate oxidation.

Degradation of myxovirus virion RNA by periodate. Extensively degraded RNA was isolated from virions of influenza virus which had been oxidized with sodium m-periodate. Similarly, although to a lesser extent, RNA isolated from periodate-treated ribonucleoprotein of influenza virus was also degraded. In contrast, influenza virus RNA, if first freed from other virion components, was not degraded by periodate oxidation.

pubmed23n0001_172

Failure of rubella virus to replicate in mosquitos.

Rubella virus failed to replicate in mosquitoes (Aedes albopictus and Culex fatigans) following parenteral inoculation. The virus demonstrated in mosquitoes tested immediately after inoculation but was not detected in insects sampled 7, 14 and 21 days postinoculation. This experiment provides further evidence that rubella is not an arbovirus, but does not invalidate its classification as a togavirus.

Failure of rubella virus to replicate in mosquitos. Rubella virus failed to replicate in mosquitoes (Aedes albopictus and Culex fatigans) following parenteral inoculation. The virus demonstrated in mosquitoes tested immediately after inoculation but was not detected in insects sampled 7, 14 and 21 days postinoculation. This experiment provides further evidence that rubella is not an arbovirus, but does not invalidate its classification as a togavirus.

pubmed23n0001_173

Treatment of severe hypertension with minoxidil.

Minoxidil in daily doses of 6 to 40 mg was administered to 11 patients with severe hypertension. Two patients died of causes unrelated to the drug and one patient withdrew from the study. Blood pressure was controlled in the remaining eight subjects, who received the drug for periods ranging from 5 to 40 months. In three patients minoxidil could subsequently be replaced by conventional antihypertensive therapy. Adverse effects of minoxidil included fluid retention (as assessed by edema and plasma volume studies), nonspecific ECG changes, hypertrichosis and conjunctival redness. Concomitant administration of diuretic and beta-adrenergic blocking agents resulted in excellent tolerance of the treatment and high patient compliance.

Treatment of severe hypertension with minoxidil. Minoxidil in daily doses of 6 to 40 mg was administered to 11 patients with severe hypertension. Two patients died of causes unrelated to the drug and one patient withdrew from the study. Blood pressure was controlled in the remaining eight subjects, who received the drug for periods ranging from 5 to 40 months. In three patients minoxidil could subsequently be replaced by conventional antihypertensive therapy. Adverse effects of minoxidil included fluid retention (as assessed by edema and plasma volume studies), nonspecific ECG changes, hypertrichosis and conjunctival redness. Concomitant administration of diuretic and beta-adrenergic blocking agents resulted in excellent tolerance of the treatment and high patient compliance.

pubmed23n0001_174

[Parotid secretion and its pharmacologically induced variants (author's transl)].

The secretion of alpha-amylase and electrolytes by the parotid gland is now well understood and similarities exist in this respect between man and animals. Many modern drugs influence parotid secretion and morphology, and these are discussed.

[Parotid secretion and its pharmacologically induced variants (author's transl)]. The secretion of alpha-amylase and electrolytes by the parotid gland is now well understood and similarities exist in this respect between man and animals. Many modern drugs influence parotid secretion and morphology, and these are discussed.

pubmed23n0001_175

Acute renal failure.

Acute renal failure is a life-threatening situation for which satisfactory treatment is currently available. Death is usually related to concomitant infection rather than uremia. Prompt recognition and treatment of underlying factors that predispose to renal insufficiency can frequently prevent the development of intrinsic acute renal failure.

Acute renal failure. Acute renal failure is a life-threatening situation for which satisfactory treatment is currently available. Death is usually related to concomitant infection rather than uremia. Prompt recognition and treatment of underlying factors that predispose to renal insufficiency can frequently prevent the development of intrinsic acute renal failure.

pubmed23n0001_176

Analysis of pesticides by chemical derivatization. I. a new procedure for the formation of 2-chloroethyl esters of ten herbicidal acids.

A new esterification procedure was developed to form 2-chloroethyl esters for 10 common herbicidal acids, using dicyclohexyl carbodiimide and 2-chloroethanol. This reagent was found to be more desirable than the commonly used boron trichloride/2-chloroethanol reagent. Maximum esterification was given for picloram at a lower reaction temperature with a shorter reaction time. Also, maximum esterification of dicamba and 2,3,6-TBA was achieved. The boron trichloride/2-chlorethanol gave poor or little esterification with these 2 acids under the various conditions investigated. The effect of solvents on esterification with the 2 reagents was also explored.

Analysis of pesticides by chemical derivatization. I. a new procedure for the formation of 2-chloroethyl esters of ten herbicidal acids. A new esterification procedure was developed to form 2-chloroethyl esters for 10 common herbicidal acids, using dicyclohexyl carbodiimide and 2-chloroethanol. This reagent was found to be more desirable than the commonly used boron trichloride/2-chloroethanol reagent. Maximum esterification was given for picloram at a lower reaction temperature with a shorter reaction time. Also, maximum esterification of dicamba and 2,3,6-TBA was achieved. The boron trichloride/2-chlorethanol gave poor or little esterification with these 2 acids under the various conditions investigated. The effect of solvents on esterification with the 2 reagents was also explored.

pubmed23n0001_177

Local control of pulmonary resistance and lung compliance in the canine lung.

Local control of pulmonary resistance and lung compliance was studied in the in situ left lower lobe of the canine lung. Recirculation of blood through the lobe while the Pco2 of the ventilatory gas was varied resulted in an increase in resistance and a decrease in compliance only when the pulmonary venous pH was greater than 7.42. Alternating sodium bicarbonate and lactic acid infusion while alveolar Pco2 was maintained below 5 mmHg demonstrated the dependence of the hypocapnic response on the acid-base status of the blood perfusing the respiratory airways. The increase in resistance and decrease in compliance observed at a pulmonary venous pH of 7.64 was comparable to that observed after lobar pulmonary artery occlusion. Varying degrees of hypoxia did not significantly affect bronchomotor tone, nor was the bronchoconstriction following lobar pulmonary artery occlusion affected by the hypoxia. Vagal stimulation superimposed on a stepwise increase in pulmonary venous pH from 7.32 to 7.62 resulted in an increase in resistance which paralleled the increase in resistance when pulmonary venous pH alone was increased. Compliance was not significantly affected by vagal stimulation at any level of pulmonary venous pH.

Local control of pulmonary resistance and lung compliance in the canine lung. Local control of pulmonary resistance and lung compliance was studied in the in situ left lower lobe of the canine lung. Recirculation of blood through the lobe while the Pco2 of the ventilatory gas was varied resulted in an increase in resistance and a decrease in compliance only when the pulmonary venous pH was greater than 7.42. Alternating sodium bicarbonate and lactic acid infusion while alveolar Pco2 was maintained below 5 mmHg demonstrated the dependence of the hypocapnic response on the acid-base status of the blood perfusing the respiratory airways. The increase in resistance and decrease in compliance observed at a pulmonary venous pH of 7.64 was comparable to that observed after lobar pulmonary artery occlusion. Varying degrees of hypoxia did not significantly affect bronchomotor tone, nor was the bronchoconstriction following lobar pulmonary artery occlusion affected by the hypoxia. Vagal stimulation superimposed on a stepwise increase in pulmonary venous pH from 7.32 to 7.62 resulted in an increase in resistance which paralleled the increase in resistance when pulmonary venous pH alone was increased. Compliance was not significantly affected by vagal stimulation at any level of pulmonary venous pH.

pubmed23n0001_178

Inactivation of citrate lyase from Rhodopseudomonas gelatinosa by a specific deacetylase and inhibition of this inactivation by L-(+1-glutamate.

A previously unrecognized enzyme, citrate lyase deacetylase, has been purified about 140-fold from cell extracts of Rhodopseudomonas gelatinosa. It catalyzed the conversion of enzymatically active acetyl-S-citrate lyase into the inactive HS-form and acetate. The enzyme exhibited an optimal rate of inactivation at pH 8.1. Because of the instability of acetyl-S-citrate lyase at acidic and alkaline pH values, all assays were carried out at pH 7.2, where the spontaneous hydrolysis of the acetyl-S-citrate lyase was negligible and deacetylase showed 70% of the activity at pH 8.1. The apparent Km value for citrate lyase was 10(-7) M at pH 7.2 and 30 C. The activity of the deacetylase was restricted to the citrate lyase from R. gelatinosa. The corresponding lyases from Enterobacter aerogenes (formerly Klebsiella aerogenes) and Streptococcus diacetilactis were not deacetylated; likewise, thioesters such as acetyl-S coenzyme A, acetoacetyl-S coenzyme A, and N-acetyl-S-acetyl-cysteamine were also not hydrolyzed. Citrate lyase deacetylase was present in very small amounts in cells of R. gelatinosa grown with acetate or succinate; it was induced by citrate along with the citrate lyase. L-(+)-Glutamate strongly inhibited the deacetylase. Fifty percent inhibition was obtained at a concentration of 1.4 X 10(-4) L-(+)-glutamate. D-(-)-Glutamate, alpha-ketoglutarate, L-alpha-hydroxyglutarate, L-(-)-proline, and other metabolites were less effective.

Inactivation of citrate lyase from Rhodopseudomonas gelatinosa by a specific deacetylase and inhibition of this inactivation by L-(+1-glutamate. A previously unrecognized enzyme, citrate lyase deacetylase, has been purified about 140-fold from cell extracts of Rhodopseudomonas gelatinosa. It catalyzed the conversion of enzymatically active acetyl-S-citrate lyase into the inactive HS-form and acetate. The enzyme exhibited an optimal rate of inactivation at pH 8.1. Because of the instability of acetyl-S-citrate lyase at acidic and alkaline pH values, all assays were carried out at pH 7.2, where the spontaneous hydrolysis of the acetyl-S-citrate lyase was negligible and deacetylase showed 70% of the activity at pH 8.1. The apparent Km value for citrate lyase was 10(-7) M at pH 7.2 and 30 C. The activity of the deacetylase was restricted to the citrate lyase from R. gelatinosa. The corresponding lyases from Enterobacter aerogenes (formerly Klebsiella aerogenes) and Streptococcus diacetilactis were not deacetylated; likewise, thioesters such as acetyl-S coenzyme A, acetoacetyl-S coenzyme A, and N-acetyl-S-acetyl-cysteamine were also not hydrolyzed. Citrate lyase deacetylase was present in very small amounts in cells of R. gelatinosa grown with acetate or succinate; it was induced by citrate along with the citrate lyase. L-(+)-Glutamate strongly inhibited the deacetylase. Fifty percent inhibition was obtained at a concentration of 1.4 X 10(-4) L-(+)-glutamate. D-(-)-Glutamate, alpha-ketoglutarate, L-alpha-hydroxyglutarate, L-(-)-proline, and other metabolites were less effective.

pubmed23n0001_179

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.

Novel type of murein transglycosylase in Escherichia coli. The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.

pubmed23n0001_180

Formation and cleavage of 2-keto-3-deoxygluconate by 2-keto-3-deoxygluconate aldolase of Aspergillus niger.

2-Keto-3-deoxygluconate aldolase of Aspergillus niger, an enzyme that has not been reported previously, was purified 468-fold. Maximal activity was obtained at pH 8.0 and 50 C. The enzyme exhibited relative stereochemical specificity with respect to glyceraldehyde. The Km values for 2-keto-3-deoxygluconate, glyceraldehyde, and pyruvate were 10, 13.3, and 3.0 mM, respectively. The effects of some compounds and inhibitors on enzyme activity were examined. Stability of the enzyme under different conditions was investigated. The equilibrium constant was about 0.33 X 10(-3) M.

Formation and cleavage of 2-keto-3-deoxygluconate by 2-keto-3-deoxygluconate aldolase of Aspergillus niger. 2-Keto-3-deoxygluconate aldolase of Aspergillus niger, an enzyme that has not been reported previously, was purified 468-fold. Maximal activity was obtained at pH 8.0 and 50 C. The enzyme exhibited relative stereochemical specificity with respect to glyceraldehyde. The Km values for 2-keto-3-deoxygluconate, glyceraldehyde, and pyruvate were 10, 13.3, and 3.0 mM, respectively. The effects of some compounds and inhibitors on enzyme activity were examined. Stability of the enzyme under different conditions was investigated. The equilibrium constant was about 0.33 X 10(-3) M.

pubmed23n0001_181

Chemotaxis of a motile Streptococcus toward sugars and amino acids.

A motile Streptococcus was isolated and its chemotactic behavior toward sugars and amino acids was studied. Motility was optimal in the presence of an exogenous energy source and a nonionic detergent, e.g., Tween 80 or Brij-36. Both glucose and pyruvate could serve as energy source. Chemotaxis toward leucine was optimal at pH 7 to 8.5 and a temperature between 30 and 37 C. The Streptococcus showed a chemotactic response toward a variety of sugars. All commonly occurring L-amino acids, except alanine, asparagine, aspartate, glutamate, arginine, and lysine, were attractants. From concentration response curves the thresholds, peak concentrations, and optimal responses were determined.

Chemotaxis of a motile Streptococcus toward sugars and amino acids. A motile Streptococcus was isolated and its chemotactic behavior toward sugars and amino acids was studied. Motility was optimal in the presence of an exogenous energy source and a nonionic detergent, e.g., Tween 80 or Brij-36. Both glucose and pyruvate could serve as energy source. Chemotaxis toward leucine was optimal at pH 7 to 8.5 and a temperature between 30 and 37 C. The Streptococcus showed a chemotactic response toward a variety of sugars. All commonly occurring L-amino acids, except alanine, asparagine, aspartate, glutamate, arginine, and lysine, were attractants. From concentration response curves the thresholds, peak concentrations, and optimal responses were determined.

pubmed23n0001_182

Phospholipase D activity of gram-negative bacteria.

A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.

Phospholipase D activity of gram-negative bacteria. A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.

pubmed23n0001_183

Pyridine nucleotide-linked oxidation of methanol in methanol-assimilating yeasts.

An alcohol dehydrogenase linked to nicotinamide adenine dinucleotide and requiring glutathione has been isolated and partially purified from two methanol-assimilating yeasts. It differs from previously described methanol-oxidizing enzymes in pH optima, electron acceptor specificity, substrate specificity, inhibition pattern, and stability.

Pyridine nucleotide-linked oxidation of methanol in methanol-assimilating yeasts. An alcohol dehydrogenase linked to nicotinamide adenine dinucleotide and requiring glutathione has been isolated and partially purified from two methanol-assimilating yeasts. It differs from previously described methanol-oxidizing enzymes in pH optima, electron acceptor specificity, substrate specificity, inhibition pattern, and stability.

pubmed23n0001_184

Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli.

The normal hut (histidine utilization) operons, as well as those with mutations affecting the regulation of their expression, of Salmonella typhimurium were introduced on an F' episome into cells of S. typhimurium and Klebsiella aerogenes whose chromosomal hut genes had been deleted and into cells of Escherichia coli, whose chromosome does not carry hut genes. The episomal hut operons respond in a manner very similar to induction and catabolite repression in all three organisms. The small differences found reflect both different abilities to take up inducers from the medium and different degrees of catabolite repression exerted by glucose.

Expression of the hut operons of Salmonella typhimurium in Klebsiella aerogenes and in Escherichia coli. The normal hut (histidine utilization) operons, as well as those with mutations affecting the regulation of their expression, of Salmonella typhimurium were introduced on an F' episome into cells of S. typhimurium and Klebsiella aerogenes whose chromosomal hut genes had been deleted and into cells of Escherichia coli, whose chromosome does not carry hut genes. The episomal hut operons respond in a manner very similar to induction and catabolite repression in all three organisms. The small differences found reflect both different abilities to take up inducers from the medium and different degrees of catabolite repression exerted by glucose.

pubmed23n0001_185

Regulation of the hut operons of Salmonella typhimurium and Klebsiella aerogenes by the heterologous hut repressors.

In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.

Regulation of the hut operons of Salmonella typhimurium and Klebsiella aerogenes by the heterologous hut repressors. In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.

pubmed23n0001_186

Transport of molybdate by Clostridium pasteurianum.

The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated. Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate. The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0). The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively. Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki [SO42-] is 3.0 X 10(-5) M; apparent Ki [WO42-] is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect. Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable. Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-.

Transport of molybdate by Clostridium pasteurianum. The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated. Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate. The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0). The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively. Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki [SO42-] is 3.0 X 10(-5) M; apparent Ki [WO42-] is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect. Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable. Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-.

pubmed23n0001_187

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium. The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.

pubmed23n0001_188

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus. A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.

pubmed23n0001_189

Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae.

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.

Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae. The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.

pubmed23n0001_190

D-Lactate dehydrogenase of Peptostreptococcus elsdenii.

D-Lactate dehydrogenase has been purified to near homogeneity from Peptostreptococcus elsdenii. As isolated, the enzyme contains flavine adenine dinucleotide and a tightly bound metal cofactor. Inactivation by ortho-phenanthroline occurs in two steps and is partially blocked by D-lactate. Reactivation by divalent metal ions occurs, with divalent zinc being the most effective. When ferricyanide is used as the electron acceptor, D-lactate has an apparent K0.5 of 3.3 M0.46; its binding is negatively cooperative with a Hill coefficient of 0.46. Replacement of ferricyanide by the other components of the electron transport system yields hyperbolic kinetics with an apparent Km for D-lactate of 26 mM. The apparent Km for ferricyanide is 2.2 X 10(-4) M. Phosphate and pyrophosphate compounds stimulate the D-lactate:ferricyanide activity. These properties suggest that interaction of this enzyme with other electron transport proteins in the chain may enhance D-lactate binding and, hence, the rate of electron transport.

D-Lactate dehydrogenase of Peptostreptococcus elsdenii. D-Lactate dehydrogenase has been purified to near homogeneity from Peptostreptococcus elsdenii. As isolated, the enzyme contains flavine adenine dinucleotide and a tightly bound metal cofactor. Inactivation by ortho-phenanthroline occurs in two steps and is partially blocked by D-lactate. Reactivation by divalent metal ions occurs, with divalent zinc being the most effective. When ferricyanide is used as the electron acceptor, D-lactate has an apparent K0.5 of 3.3 M0.46; its binding is negatively cooperative with a Hill coefficient of 0.46. Replacement of ferricyanide by the other components of the electron transport system yields hyperbolic kinetics with an apparent Km for D-lactate of 26 mM. The apparent Km for ferricyanide is 2.2 X 10(-4) M. Phosphate and pyrophosphate compounds stimulate the D-lactate:ferricyanide activity. These properties suggest that interaction of this enzyme with other electron transport proteins in the chain may enhance D-lactate binding and, hence, the rate of electron transport.

pubmed23n0001_191

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.

pubmed23n0001_192

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans.

Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.

Isolation and purification of Flavobacterium alpha-1,3-glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans. Studies were made on the physical and chemical properties of polysaccharides synthesized by cell-free extracts of Streptococcus mutans, Streptococcus sanguis, and Streptococcus sp. and their susceptibilities to dextranases. Among the polysaccharides examined, insoluble glucans were rather resistant to available dextranase preparations, and the insoluble, sticky glucan produced by S. mutans OMZ 176, which could be important in formation of dental plaques, was the most resistant. By enrichment culture of soil specimens, using OMZ 176 glucans as the sole carbon source, an organism was isolated that produced colonies surrounded by a clear lytic zone on opaque agar plates containing the OMZ 176 glucan. The organism was identified as a strain of Flavobacterium and named the Ek-14 bacterium. EK-14 bacterium was grown in Trypticase soy broth, and an enzyme capable of hydrolyzing the OMZ 176 glucan was concentrated from the culture supernatant and purified by negative adsorption on a diethylaminoethyl-cellulose (DE-32) column and gradient elution chromatography with a carboxymethyl-cellulose (CM-32) column. The enzyme was a basic protein with an isoelectric point of pH 8.5 and molecular weight of 65,000. Its optimum pH was 6.3 and its optimal temperature was 42 C. The purified enzyme released 11% of the total glucose residues of the OMZ 176 glucan as reducing sugars and solubilized about half of the substrate glucan. The products were found to be isomaltose, nigerose, and nigerotriose, with some oligosaccharides. The purified enzyme split the alpha-1,3-glucan endolytically and was inactive toward glucans containing alpha-1,6, alpha-1,4, beta-1,3, beta-1,4, and/or beta-1,6 bonds as the main linkages.

pubmed23n0001_193

Production, purification, and characterization of an extracellular chitosanase from Streptomyces.

The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall.

Production, purification, and characterization of an extracellular chitosanase from Streptomyces. The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall.

pubmed23n0001_194

Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures.

After mutagenesis, surviving yeast cells are grown on plates at 25 C and later exposed to 37 C. The plates are then overlaid with a soft agar containing p-nitrophenylphosphate at pH 9.7. Lysed cells liberate alkaline phosphatase which gives rise to a yellow color on and around colonies.

Simple and sensitive procedure for screening yeast mutants that lyse at nonpermissive temperatures. After mutagenesis, surviving yeast cells are grown on plates at 25 C and later exposed to 37 C. The plates are then overlaid with a soft agar containing p-nitrophenylphosphate at pH 9.7. Lysed cells liberate alkaline phosphatase which gives rise to a yellow color on and around colonies.

pubmed23n0001_195

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme. A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.

pubmed23n0001_196

Rapid radioimmunoassay for guanosine 3',5'-cyclic monophosphate using tritiated ligand.

A radioimmunoassay procedure for guanosine 3',5'-cyclic monophosphate (CGMP) is described. The procedure is based on competitive binding between [3H]CGMP and non-radioactive CGMP, with separation of bound and unbound CGMP by Millipore filtration. The binding reaction showed very high specificity to CGMP, had a broad pH optimum, and reached equilibrium within a short time. A simple procedure for the pruification of assay samples using Dowex AG 50W-X2 resin is also described. CGMP contents in urine samples were assayed without purification. Injection of glucagon into healthy human volunteers resulted in a small but significant reduction in urinary CGMP level, whereas CAMP excretion increased dramatically.

Rapid radioimmunoassay for guanosine 3',5'-cyclic monophosphate using tritiated ligand. A radioimmunoassay procedure for guanosine 3',5'-cyclic monophosphate (CGMP) is described. The procedure is based on competitive binding between [3H]CGMP and non-radioactive CGMP, with separation of bound and unbound CGMP by Millipore filtration. The binding reaction showed very high specificity to CGMP, had a broad pH optimum, and reached equilibrium within a short time. A simple procedure for the pruification of assay samples using Dowex AG 50W-X2 resin is also described. CGMP contents in urine samples were assayed without purification. Injection of glucagon into healthy human volunteers resulted in a small but significant reduction in urinary CGMP level, whereas CAMP excretion increased dramatically.

pubmed23n0001_197

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions. 1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.

pubmed23n0001_198

Electrophoretic investigations of the acid conformational change of alpha-lactalbumin.

In order to clarify how the electrophoretic behavior reflects the conformational transition of globular proteins, moving boundary electrophoresis was applied to analysis of the acid conformational change of alpha-lactalbumin. The appearance of only a single electrophoretic boundary in the transition region of the protein suggests a very rapid transition with a half-time estimated to be smaller than 7 min on the basis of the theory of isomerizing systems in electrophoresis. The transition is clearly reflected in the dependence of the mobility on the protein net charge, which shows a sigmoidal curve closely similar to that obtained by a Linderstrøm-Lang pH-tritration plot for the carboxyl groups of alpha-lactalbumin. It was also concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein. All results obtained by electrophoresis were also supported by the results of pH-jump studies, analytical gel chromatography, and CD measurements.

Electrophoretic investigations of the acid conformational change of alpha-lactalbumin. In order to clarify how the electrophoretic behavior reflects the conformational transition of globular proteins, moving boundary electrophoresis was applied to analysis of the acid conformational change of alpha-lactalbumin. The appearance of only a single electrophoretic boundary in the transition region of the protein suggests a very rapid transition with a half-time estimated to be smaller than 7 min on the basis of the theory of isomerizing systems in electrophoresis. The transition is clearly reflected in the dependence of the mobility on the protein net charge, which shows a sigmoidal curve closely similar to that obtained by a Linderstrøm-Lang pH-tritration plot for the carboxyl groups of alpha-lactalbumin. It was also concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein. All results obtained by electrophoresis were also supported by the results of pH-jump studies, analytical gel chromatography, and CD measurements.

pubmed23n0001_199

Studies on cathepsins of rat liver lysosomes. II. Comparative studies on multiple forms of cathepsin A.

The multiple forms of cathepsin A (AI, AII, and AIII) purified from the lysosome fraction of rat liver by Sephadex G-200 and DEAE-Sephadex chromatographies were studied comparatively. Forms AI, AII and AIII were stable between pH 3.0 and 5.5, and had pH optima for CBZ-Glu-Phe at 5.6, 5.8, and 5.9, respectively. These activities were rapidly lost on heating above 60 degrees. Their isoelectric points were at 4.7, 4.8, and 4.9, and the Michaelis constants for CBZ-Glu-Phe were calculated as 10, 6.6, and 4.2 X 10(-4)M, respectively. Activity was inhibited by Ag+, Au3+, Hg2+, iodine, and p-chloromercuribenzoate (PCMB). Diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), toluenesuffonyl fluoride (TSF), and sodium dodecyl sulfate (SDS) were inhibitory at a concentration of 10(-3)M. Soybean trypsin inhibitor, pepstatin, leupeptins, and antipain were not inhibitory, while chymostatin caused slight inhibition. No distinct difference was observed in the effects of these compounds on the multiple forms of cathepsin A despite differences in the molecular weights of these forms (100,000, 200,000, and 420,000, respectively). In immuno-diffusion analysis, cathepsin AI, AII, and AIII which had been treated with EDTA, dithiothreitol, PCMB, and a high concentration of NaCl, gave the same precipitin patterns as the untreated enzymes, but treatment with 6 M urea caused a slight alteration of the pattern. After SDS-treatment (50 mM or more), the precipitin lines of these multiple forms fused and gave a single, identical line. This suggests that the different forms of the cathepsin A are all composed of subunits which are immunologically identical or closely related, and that the subunits are mainly bound by hydrophobic forces. This conclusion is supported by results obtained by poliacrylamide gel electrophoresis in the presence of SDS.

Studies on cathepsins of rat liver lysosomes. II. Comparative studies on multiple forms of cathepsin A. The multiple forms of cathepsin A (AI, AII, and AIII) purified from the lysosome fraction of rat liver by Sephadex G-200 and DEAE-Sephadex chromatographies were studied comparatively. Forms AI, AII and AIII were stable between pH 3.0 and 5.5, and had pH optima for CBZ-Glu-Phe at 5.6, 5.8, and 5.9, respectively. These activities were rapidly lost on heating above 60 degrees. Their isoelectric points were at 4.7, 4.8, and 4.9, and the Michaelis constants for CBZ-Glu-Phe were calculated as 10, 6.6, and 4.2 X 10(-4)M, respectively. Activity was inhibited by Ag+, Au3+, Hg2+, iodine, and p-chloromercuribenzoate (PCMB). Diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), toluenesuffonyl fluoride (TSF), and sodium dodecyl sulfate (SDS) were inhibitory at a concentration of 10(-3)M. Soybean trypsin inhibitor, pepstatin, leupeptins, and antipain were not inhibitory, while chymostatin caused slight inhibition. No distinct difference was observed in the effects of these compounds on the multiple forms of cathepsin A despite differences in the molecular weights of these forms (100,000, 200,000, and 420,000, respectively). In immuno-diffusion analysis, cathepsin AI, AII, and AIII which had been treated with EDTA, dithiothreitol, PCMB, and a high concentration of NaCl, gave the same precipitin patterns as the untreated enzymes, but treatment with 6 M urea caused a slight alteration of the pattern. After SDS-treatment (50 mM or more), the precipitin lines of these multiple forms fused and gave a single, identical line. This suggests that the different forms of the cathepsin A are all composed of subunits which are immunologically identical or closely related, and that the subunits are mainly bound by hydrophobic forces. This conclusion is supported by results obtained by poliacrylamide gel electrophoresis in the presence of SDS.