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Role of hyperkalemia in the metabolic acidosis of isolated hypoaldosteronism. We studied the relative importance of hyperkalemia and mineralocorticoid deficiency in the metabolic acidosis of a patient with proved isolated hyporeninemic hypoaldosteronism and moderate kidney failure. The hyperkalemia and acidosis were severe in relation to the slight azotemia. Despite the systemic acidosis and urinary pH of 4.9, urinary ammonium excretion was distinctly blunted. Correction of the hyperkalemia by potassium-sodium exchange resin alone resolved the acidosis and restored the previously diminished urinary ammonium excretion to normal. Administration of mineralocorticoids only partially corrected the hyperkalemia and the acidosis. Hyperkalemia by itself, rather than hypoaldosteronism per se, caused the acidosis in this patient. Hyperkalemia apparently suppresses urinary ammonium excretion and thus interferes with urinary acidification.
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Isolation of Penicillium corylophium Dierckx from acid mine water and its optimal growth on hydrocarbons at acid pH. Penicillium corylophilium Dieckx was isolated from sludge collected at the interface of an aqueous, copper-bearing leachate and an organic, kerosene based, ion exchange solvent. The organism assimilated kerosene and various straight chain and cyclic hydrocarbons including dodecane, hexadecane, octadecane, toluene, benzene, and cyclohexane. Assimilation of kerosene and hexadecane was optimal at pH 2 and was stimulated by yeast extract.
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The effect of microbial mycolytic agents on Trichophyton rubrum. Chitinolytic microorganisms isolated from forest soil and from healthy gypsy moth larvae (Porthetria dispar (L.) were screened for their ability to lyse Trichophyton rubrum mycelia. A few of these isolates were mycolytic on both autoclaved and on actively growing, intact, T. rubrum mycelia. Supernatants from these isolates, utilizing live T. rubrum as the sole carbon source, showed the same mycolytic ability. Assays of the supernatants for enzymatic activity revealed exocellular, stable enzymes that releases reducing substances including N-acetylglucosamine from the mycelia.
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Models for metal ion function in carbonic anhydrase. A model catalyst is described which has properties in common with carbonic anhydrase. The model demonstrates the availability of a mechanism, previously only hypothetical, for the action of the enzyme. It also shows, however, that this mechanism alone is not adequate to produce the high activity of the enzyme.
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[Regulation of lipid synthesis in animal organs]. Studies were made of the mechanisms regulating the quantity and catalytic efficiency of hepatic acetyl coenzyme A carboxylase, which plays a critical role in the control of fatty acid biosynthesis. The microsomal enzyme system responsible for the formation of phosphatidic acid, the initial step in glycerolipid biosynthesis, was resolved into two component enzymes. The acyl-donor specificities of these and other acyltransferases account for the asymmetric fatty acid distribution in naturally occurring glycerolipids.
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Comparison of properties between virulent and attenuated strains of transmissible gastroenteritis virus. Strains of transmissible gastroenteritis (TGE) virus possessing different pathogenicity were examined for stability to digestive enzymes and acid, and growth at various temperatures. In growth experiments, virus titer obtained at 37 degrees C were about equal between attenuated and virulent strains, but titers attained by the attenuated strain were higher at 30 degrees C. The attenuated virus multiplied at 28 degrees C, but the virulent virus did not at this temperature. The virulent virus was significantly stable to trypsin and pepsin, but the attenuated virus was inactivated rapidly by these proteolytic enzymes. No significant differences were observed in stability to acid between the attenuated and virulent strains. At different pH, both lost their infectivity more rapidly at 37 degrees C than at 22 degrees C.
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Growth of Ibaraki virus in suspension culture of HmLu-1 cells. The established hamster lung cell line, HmLu-1 cells could grow in a suspended state. The initial cell count, 40 X 10(4)/ml, increased to 200 X 10(4)/ml on the 4th day of culture. The suspension culture of HmLu-1 cells was proved satisfactory for propagation of Ibaraki virus. The viral titer reached a maximum of 10(6.75) TCID50/0.1 ml. The input multiplicity ranging from 0.003 to 3.0 exerted no influence on the final yield of the virus. The optimal pH value of initial culture ranged from 6.8 to 7.6. In comparison of virus yield per cell among the suspension culture and two methods of monolayer culture in stationary and rolling condition, there was no noticeable difference in it among the three methods. The cell population per unit volume was the largest and, therefore, virus titer in the culture fluid the highest in the suspension culture of the three methods.
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Studies on the mechanism of the action of morphine on the peristalsis of guinea pig ileum in situ. The influence of some drugs on the effect of morphine on the threshold pressure required to elicit peristalsis in the guinea pig ileum in situ was studied, in order to test the hypothesis that this effect of morphine is mediated by catecholamine release. Tachyphylaxis to this effect of morphine was confirmed. Pretreatemnt with two 8 mg/kg doses of reserpine, 24 and 48 hrs before the experiment, significantly reduced the effect of morphine on the pressure threshold. The i.v. administration of 10 mg/kg dl-Dopa re-established the effect of morphine in reserpinized animals to the level of the untreated controls. Pretreatment with guanethidine (15 mg/kg) decreased and even prevented this effect of morphine. Phentolamine pretreatment (10 mg/kg) also significantly inhibited the effect of morphine. Neither DCI nor propranolol influenced this morphine effect. Pretreatment with reserpine, guanethidine or phentolamine reduced the basic pressure threshold needed to elicit peristalsis. The possibility that the decrease of local circulation induced by hypotenison would reduce the local concentration of morphine was rejected because the same doses of guanethidine or phentolamine did not modify the effect of hexamethonium given i.v. in this preparation. All these results support the idea that the effect of morphine on intestinal peristalsis is mediated by a catecholamine acting on alpha-receptors, e.g. norepinephrine.
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Effects of alpha-ketomonocarboxylic acids upon insulin secretion and metabolism of isolated pancreatic islets. In perifused isolated pancreatic islets alpha-ketoisocaproic acid (KIC) or alpha-ketocaproic acid (KC) induced a high insulin secretion rate and a steep increase of the fluorescence of reduced pyridine pyridine nucleotides [NAD(P)H] which fell again to almost prestimulatory levels 6 min after medium change. Insulin release in response to alpha-ketooctanoic (KO) acid started slowly and was accompanied by a decrease of the NAD(P)H-fluorescence trace. Beta-phenylpyruvate which is known to initiate insulin release also caused a fluorescence decrease. Alpha-keto-isovaleric (KIV) acid or pyruvate had no significant effects upon insulin secretion or NAD(P)H-fluorescence. In contrast to l-leucine, l-norleucine or l-valine did not enhance insulin release or fluorescence of NAD(P)H. KIV, alpha-keto-beta-methylvaleric acid (KMV), KIC and KC raised the production their corresponding amino acids by islet cells. From these results it is concluded that alpha-ketomonocarboxylic acids as such trigger insulin release by acting upon receptor sites which differ from those occupied by amino acids.
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The preparation and properties of nerve growth factor protein at alkaline pH. The nerve growth factor (NGF) subunit of 7S NGF was isolated by chromatography at high pH on QAE-Sephadex. It has the same specific NGF activity as betaNGF isolated at acid pH, showing that this activity is an intrinsic property of the subunit and is independent of the pathway of dissociation. Continued exposure of the NGF subunit to high pH resulted in an increase in the amount of the minor species beta2NGF and the formation of a new species, beta3NGF, of even lower isoelectric point. These two species and the original major species of the preparation, beta1, were isolated by isoelectric focusing. All three species had the same specific NGF activity, but differed in their ability to reform 7S NGF. The beta2 species was one-fifth as competent as beta1, while beta3 was unable to regenerate 7S NGF. Addition of alpha- and gamma-subunits to beta1NGF decreased the amount of NGF protein required to produce one Biological Unit of activity in the bioassay, but had no effect when added to beta3NGF. The interactions between the subunits in 7S NGF therefore determine, in part, the specific activity of the NGF subunit.
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Concanavalin A-binding glycopeptides from rat brain glycoproteins. The affinity of concanavalin A for neutral and acidic glycopeptides derived from rat brain glycoproteins was investigated by studying the inhibition of a concanavalin A-glycogen precipitation system. The neutral, mannose-rich glycopeptides obtained by column electrophoresis of the dialyzable glycopeptides that had been solubilized by proteolytic treatment of defatted brain tissue were powerful inhibitors, with an inhibitory activity 20 to 26 times that of the standard inhibitor, methyl-alpha-D-mannoside. The acidic sialoglycopeptides had activities one to nine times that of the mannoside. Therefore, both acid and neutral glycopeptides were capable of interacting with concanavalin A. The especially strong affinity of the neutral mannose-rich glycopeptides, however, enabled their retention on concanavalin A-Sepharose and subsequent elution with methyl-alpha-D-mannoside. This provided the means of separation of the acidic sialoglycopeptides from the neutral, mannose-rich glycopeptides by affinity chromatography. Glycopeptides that contain N-acetylgalactosamine are not retained by concanavalin A-Sepharose.
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The use of hybrid molecules in a study of the equilibrium between nerve growth factor monomers and dimers. The major protein in beta nerve growth factor preparations, beta1NGF, is a dimer in which both peptide chains have COOH-terminal arginine residues. Digestion of beta1NGF with carboxypeptidase B produced a dimer, beta3NGF, in which both chains lack these terminal arginine residues. Exposure of mixtures of beta1 and beta3NGF dimers to 8 M urea to produce monomers, followed by removal of urea to allow recombination, resulted in the formation of the hybrid beta2NGF, comprising one arginine-containing and one arginine-less chain, as well as the parent dimers. The amount of the three dimers formed was close to that expected from random association of monomers. Hybrid beta2NGF was also formed from mixtures of beta1 and beta3NGF where incubated at pH 2.6 to 4.5. The formation of beta2NGF has a half-time of 6 h at pH 4.0 and 4 degrees C. Its rate of formation decreased above pH 4.5, becoming minimal between pH 9.5 and pH 10.5, and increased with increasing temperature. The amount of beta2NGF formed was determined by the lowest pH to which the parent mixture was exposed, irrespective of its prior history. These data suggest that the hybrid is formed by the same mechanism in the absence and presence of the urea step. An approximate value for Kd, the equilibrium dissociation constant of the dimer equilibrium monomer equilibrium was derived. Its value was 3 - 10(-10) M at pH 4.0 and 4 degrees C. The alpha-subunit of 7S NGF decreased the rate of formation of beta2NGF not only at pHs where an alphabeta complex is stable, but also at an acid pH where no complex formation is observed by sedimentation analysis, suggesting that the present methodology offers a more sensitive probe of subunit interactions. In contrast, the gamma subunit and a number of indifferent proteins had little or no effect on the appearance of beta2NGF at the pHs studied.
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Changes in hypothalamic neurotransmitter uptake following pinealectomy, superior cervical ganglionectomy or melatonin administration to rats. Rats subjected to pinealectomy or to pinealectomy plus bilateral superior cervical ganglionectomy exhibited depressed serotonin uptake by hypothalamic synaptosomes; norepinephrine, dopamine or glutamate uptake was not affected by any of the surgical procedures. Treatment with melatonin resulted in inhibition of hypothalamic serotonin accumulation but it did not modify the uptake of norepinephrine, dopamine or glutamate. These data suggest a relationship between the pineal gland and the serotoninergic nerve endings of the hypothalamus.
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Timolol maleate (Blocadren) in the treatment of essential hypertension. The antihypertensive activity of timolol (Blocadren, FROSST-MSD), a new beta blocking agent, was assessed in a single blind crossover placebo-controlled study in 14 patients with essential hypertension. Ten patients completed the trial. The average standing pressure during the placebos periods was 168/109mmHg and during the periods on treatment with timolol the average standing pressure was 139/92mmHg. All 10 patients showed some hypotensive response. The average daily dosage was 21 mg with a range of 15-30 mg. Four patients achieved optimal levels of blood pressure by the end of one week's therapy with the maximum time required being four weeks. Timolol appears to be an effective antihypertensive agent. Side effects were insignificant.
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Effects of ritodrine hydrochloride on uterine activity and the cardiovascular system in toxemic patients. The effects of ritodrine hydrochloride were evaluated in 25 toxemic patients in active labor utilizing continuous electronic monitoring of fetal and maternal cardiovascular systems and uterine activity. Fetal scalp blood and free flowing maternal antecubital venous blood was obtained for pH, Po2, Pco2, base deficit and blood glucose determinations prior to and immediately following the study period. The initial ritodrine dose was 50 mug/min for 15 minutes. The dose was increased by 50 mug/min each 15 minutes until there was a clinically apparent reduction in uterine activity. Once this was accomplished, the infusion was maintained for 30 minutes. There was a consistent increase in the maternal heart rate (MHR) and a significant rise in fetal heart rate (FHR) late in the infusion and in the postinfusion period. There was a widening of the maternal pulse pressure mainly due to a reduction in diastolic pressure with little change in the mean blood pressure. Maternal and fetal pH decreased and base deficit increased during the study although the PO2 and PCO2 remained unchanged. Maternal and fetal blood glucose rose significantly following ritodrine infusion.
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Treatment of ovarian hyperstimulation syndrome by antihistamine. Ovarian hyperstimulation was produced by human menopausal gonadotropin and chorionic gonadotropin in rabbits. A more rapid regression of the hyperstimualted ovaries was observed in an antihistamine-treated group than in a control group. The difference in regression was found to be statistically significant. The possibility of treating the ovarian hyperstimulation syndrome by antihistamine is cited.
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High pH ammonia toxicity, and the search for life on the Jovian planets. Jovian plants have enviroments apparently suitable for the evolution of life, but nevertheless, present severe challenges to organisms. One such challenge arises from the presence of ammonia. Ammonia is an efficient biocide, its effect being dependent on pH as well as on concentration. The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural enviornments of high pH and/or ammonia concentration. Escherichia coli and Bacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, survival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is, nevertheless, two to three orders of magnitude longer than for E. coli. Our data support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.
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Gamma-glutamyltranspeptidase in the rat liver after portacaval shunt. Gamma-Glutamyltranspeptidase (GGTP) activity was studied in livers of rats submitted to an end-to-side portacaval shunt (PCS) and in developing animals. To correlate the evolution of the enzymatic activity measured in vitro, histochemical techniques were used to localize enzyme activity in liver tissue. The GGTP activity in the adult rats was low and amounted to 2.0 +/- 0.1 mumol/min/g. During fetal development the enzyme activity rose beginning on the 15th gestational day from 630 +/- 97 to 1,058 +/- 20 on the first postnatal day. Then the values declined and reached nearly adult values from the 10th postnatal day. After PCS the GGTP activity exhibited a three- to sixfold increase (130 +/- 69 to 371 +/- 131) as compared with unoperated adult controls (53 +/- 13). the highest levels corresponded to those observed between the 3rd and 5th postnatal day in the developing rats. The histochemistry of GTTP in the fetal and newborn liver showed a regular distribution of the enzyme as a fine deposit in the hepatocytes throughout the whole tissue. Ten days after birth the activity was low, at the same level as in the adult rat. In the period after PCS hepatocytes began to show signs of enzymatic activity at the periphery of the hepatic lobules, which subsequently spread through the whole lobules. The increase of GGTP activity after PCS equaled the activity found in fetal animals. That correlated well in both groups with the reappearance of histologically demonstrable enzyme activity in hepatocytes.
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Cardiovascular effects of electrical stimulation of the forebrain in the fetal lamb. Modified stereotaxic techniques were applied to fetal lambs during the latter third of gestation. Electrical stimulation in the region of the hypothalamus in 10 acute experiments was associated with three patterns of arterial blood pressure and heart rate changes: a pressor-tachycardia response; a pure tachycardia response (abolished by propranolol); and a pure bradycardia response (abolished by atropine). The pressor-tachycardia response was examined in detail in 13 chronic preparations (115-135 days of gestation at operation). The systolic arterial blood pressure increase was never greater than 35 mm Hg and was probably blunted by the large noninnervated placental circulation. This pressure increase was abolished by phentolamine and was thus mediated by stimulation of alpha-adrenergic receptors. The initial tachycardia was prevented by propranolol and was due to beta-adrenergic stimulation. The tachycardia was followed in a few seconds by a bradycardia, abolished by atropine and possibly a vagal baroreflex. The pressor-tachycardia response was accentuated in two lambs who were delivered spontaneously and were studied after birth. These studies indicate that a suprabulbar neural framework exists in the fetal lamb for influencing the cardiovascular system from as early as 90 days of gestation.
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Elevated levels of immunoglobulin E in the acute febrile mucocutaneous lymph node syndrome. Mucocutaneous lymph node syndrome (MCLS) is a newly recognized disease characterized by fever persisting for more than 5 days, an erythematous skin eruption, conjunctival congestion, dry red fissured lips, reddened tongue, palms, and soles, nonpurulent lymphadenopathy, and sometines diarrhea, arthralgia, and aseptic meningitis. Additional features may include carditis, pericarditis, aneurysmal dilation and thrombosis of coronary arteries, and sudden death. There is a striking similarity of fatal cases to infantile polyarteritis nodosa, a disease recently reported to be associated with elevated levels of serium IgE. Indeed, it is likely that MCLS represents a disease which can progress to polyarteritis nodosa in infants and young children. The paired acute and convalescent serum IgE levels of 20 subjects with acute nonfatal MCLS were studied along with 20 near-age unaffected controls from the same communities in Japan. The results indicate that most if not all subjects with MCLS in the study had an elevation of total serum IgE during the acute phase of the disease (geometric mean 157 IU/ml compared with the control value of 38 IU/ml, P = 0.005). The level appeared to reach a peak 1-2 weeks after onset and declined over the ensuing 1-2 months.
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Diminished pulmonary lecithin synthesis in acidosis: experimental findings as related to the respiratory distress syndrome. Lung slices from term fetal rats were incubated in vitro at various pH values and the rates of the two de novo pathways for lecithin biosynthesis were determined by measuring the conversion of either 14C-choline (pathway 1) or 14C-methionine (pathway 2) to the phospholipid. It was observed that the choline pathway, but not phosphatidylethanolamine methylation, is pH-sensitive with maximum rates occurring at pH levels between 7.3 and 7.5; significantly less activity was found at pH levels between 7.0 and 7.2 and at pH levels between 7.6 and 8.0. Adjustment of the pH from 7.0 to 7.4 in vitro simulating the clinical correction of acidosis by alkali infusion was found to increase the conversion of choline to lecithin to a rate approximating that observed at pH 7.4. Since lecithins are the principal phospholipid components of pulmonary surfactant, and since pathway 1 is predominantly responsible for lung lecithin synthesis, the demonstration of impaired production with reduced pH offers a biochemical explanation for the pathophysiological effects of acidosis in the respiratory distress syndrome. A comparison of pH effects on choline pathway rate with the pH profiles of pathway enzymes suggests that these effects are mediated by the catalysts of lecithin synthesis.
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pH and bicarbonate excretion in the rat parotid gland as a function of salivary rate. The bicarbonate concentration in rat parotid saliva increases with increasing flow rates and approximates plasma values at highest salivation. At lowest flow rates the bicarbonate concentration in the secretory fluid markedly exceeds the plasma levels. Intravenous administration of acetazolamide has no influence on the bicarbonate excretion of the parotid gland. Following retrograde application of acetazolamide into the gland duct the concentrations of both bicarbonate and sodium are elevated. The potassium concentrations in final saliva exceed 70 mEq/l at flow rates below 5 mul/min g gland weight. With increasing flow rates a precipitous decrease in potassium concentration below 10 mEq/l occurs. With further increase in flow rate the potassium concentration remains unchanged. The sodium concentrations increased with augmented salivation rate. At lowest flow rates the sodium concentrations showed an increase of modest degree. Our findings can best be explained by the existence of two independent ductular mechanism: a) bicarbonate reabsorption probably in the striated ducts of the parotid gland; b) secretion of potassium with concomitand secretion of bicarbonate in the main excretory duct.
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H+ transport and membrane-bound HCO - 3 ATPase in salivary duct epithelium. An ATPase stimulated by HCO - ions and other oxybases and inhibited by SCN- has been found in main excretory duct of rat submaxillary gland, a tissue, capable of actively secreting HCO - 3 ions. No such ATPase was found in the rabbit duct, which normally does not secrete HCO - 3. The HCO - 3 ATPase was localized in the plasma membrane fraction of the homogenate, as evidenced by the marker 5'-nucleotidase. The activities of the HCO - 3 ATPase increased in metabolic alkalosis and decreased in metabolic acidosis in parallel to secretion of HCO - 3 and K+ ions by the duct epithelium. These findings provide further evidence that the membrane-bound HCO - 3 ATPase is involved in active H+/HCO - 3 transport.
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Effects of cyanide and doxapram during hypothermia. The ventilatory responses, blood gases and acid-base status to intravenous injections of KCN and doxapram hydrochloride were studied in anesthetized dogs during normothermia and at two levels of hypothermia. In the normothermic animal, KCN evoked significant elevations of minute and alveolar ventilations. For the mildly hypothermic (32-33 degrees C) dog, minute and alveolar ventilations were proportionally greater than for normothermia. Bolus infusions of KCN to deeply hypothermic dogs (28-29 degrees C) elicited larger and nearly similar increases of minute and alveolar ventilations as compared, respectively, with normothermia and mild hypothermia. Compared to their controls, injections of doxapram during normothermia, mild and deep hypothermia augmented VE 43.3%, 63.6% and 31.5%, respectively. With doxapram there was a feeble increase in alveolar ventilation. These results demonstrate that the peripheral (arterial) chemoreceptors preserve the capacity to respond to stimuli given acutely while lowering core temperature and in some circumstances this capacity is even enhanced as compared to normothermia.
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Effects of cyanide and doxapram during panting. The ventilatory responses, blood gases and acid-base status to intravenous injections of KCN and doxapram hydrochloride were studied in anesthetized dogs during normothermia and thermally induced panting. In the normothermic animal, KCN evoked elevation of VE (154.7%), VT (70.1%), f (48.3%, PaO2 (12.1%) and pH (0.098 units), while PaCO2 diminished by 9.7 mm Hg. During panting, KCN infusions resulted in increases of VE (24.5%), VT (46.6%), PaO2 (3.9%) and pH (0.034 units), while f decreased (10.1%). Bolus injections of doxapram during normothermia increased VE (32.6%), VT (18.8%) and f (17.1%). During panting VE, VT and f increased by 18.0%, 18.2% and 1.5%, respectively. These results demonstrate that the peripheral (arterial) chemoreceptors preserve the capacity to react to acute chemical stimuli in animals in which the thermal stimuli override the normal chemical control of respiration in order to control body temperature, and that this reaction contributes to the integrated respiratory drive.
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The effect of alpha-adrenolytics on the levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in rat's brain. Alpha-Adrenolytics (ABA)-phenoxybenzamine, phentolamine and aceperone-increase 5-hydroxyindoleacetic (5-HIAA) acid content in rat's brain. At the same time, these compounds either increase or do not affect 5-hydroxytryptamine (5-HT) level. Moreover, they potentiate L-dopa and reserpine-induced increase in 5-HIAA level and antagonized clonidine-induced decrease in 5-HIAA content. The experiments with probenecid indicate that the observed increase in 5-HIAA does not result from the deficient elimination of the metabolite from brain. The increase in 5-HT turnover in brain, due to ABA, is suggested.
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The central action of drugs influencing beta-adrenergic receptor. part V. The interaction of drugs affecting beta-adrenergic receptor in motility test. Propranolol, alprenolol and sotalol given intraventricularly (ivc) to rats reduce depressing effects of isoprenaline (IPS) and antagonize stimulating effects of noradrenaline (NA). Phentolamine given by the same route of administration does not affect IPS action.
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Combating diabetic ketoacidosis and other hyperglycemic-ketoacidotic syndromes. Diabetic ketoacidosis is an acute medical emergency that requires immediate diagnosis and treatment. Diagnosis may be established rapidly by measurement of urinary glucose and ketones, arterial blood pH and blood gases, and serum ketones. Rapid infusion of large volumes of fluids and electrolytes, together with continuous infusion of low doses of insulin, provides effective restoration of fluid and electrolyte balance and correction of metabolic derangements. Hyperosmolar nonketotic coma is characterized by marked hyperglycemia in the absence of ketoacidosis and occurs usually in patients with mild adult-onset diabetes. Symptoms develop more slowly than in diabetic ketoacidosis. Treatment is the same for both conditions. In alcoholic ketoacidosis, hyperketonemia is present without hyperglycemia. The syndrome differs from diabetic ketoacidosis in that blood glucose levels are lower and glycosuria is absent. Treatment consists of intravenous administration of dextrose in water and, if necessary, of sodium bicarbonate. Insulin administration usually is not necessary.
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The effect of alpha, alpha1-dipyridyl on noradrenaline, dopamine and 5-hydroxytryptamine levels and on dopamine-beta-hydroxylase activity in brain. The effects of alpha, alpha1-dipyridyl (DP) on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) levels in rat and mouse brain and on dopamine-beta-hydroxylase (DbetaH) activity in rat brain have been studied, DP decreases NA level and inhibits DbetaH activity in a dose dependent manner, without affecting DA or 5-HT levels.
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[Current conception of the Bohr effect]. The molecular mechanism of the Bohr effect is explained according to the molecular model proposed by Perutz et al. The Bohr effect is due to changes in the pK of specific carboxyl and amino groups of the four globin chains following the transition between the deoxy and oxy conformations of the molecule. Carbon dioxide binds to the N terminal valine of the 4 monomers to form carbamino compounds. This carbaminoformation depends upon pH, PCO2 and predominates on deoxygenated haemoglobin. It is lowered when O2 binds to the heme groups (O2 linked carbamino compounds). Through the carbamino compounds Carbon dioxide lowers both the affinity of haemoglobin for O2 and the Bohr effect. Diphosphoglycerate also binds to the haemoglogin molecule. This organophosphate lowers the affinity for O2 but increases the Bohr effect. In whole blood, the Bohr effect is therefore dependent upon pH, O2 saturation, PCO2 and DPG concentration into the red blood cells.
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[Study of P50 in patients under continuous O2 inhalation and during chronic respiratory acidosis]. In vitro, the affinity of Hb for O2 depends on pH and capnia by the intermediate of the 2-3 DPG level, the concentration of which lowers in the case of acidosis and hypercapnia. Thus, an increase in the affinity results, but while Bohr's effect is immediate, on the contrary the 2-3 DPG effect is slow. Authors have verified the importance of this modification by studying the affinity of Hb for O2 thanks to the P50 technique in 15 normal non-smokers subjects and in 10 subjects with compensated or not respiratory acidosis but normally saturated thanks to continuous O2 administration.
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Colorimetric determination of bromisoval and carbromal. New colorimetric procedure for determination of bromisoval and carbromal is based on the hydroxamation reaction and colour developing after addition of Fe(ClO4)3. The method is employed for the estimation of both compounds in substances and in tablets.
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[Effect of cultivation conditions on the synthesis of citric and isocitric acids in Candida lipolytica on hexadecane medium]. The influence of aeration, pH and iron concentration on the growth of yeast C. lipolytica 704 on the hexadecane medium and on the synthesis of citric and isocitric acids was investigated. The yeast synthesized citric acids actively during intensive aeration. The acid formation was strongly dependent on the medium acidity: pH 6.0 was most favourable for the synthesis of citric acids. The Fe concentration influenced significantly the ratio of the acids synthesized. At a low concentration of iron (0.005 mg Fe/l) equal amounts of citrate and isocitrate were formed; at an increased concentration isocitrate was in predominant formation.
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[Study of inosine transformation into 5'-inosinic acid by the culture of Pseudomonas trifoli]. The transformation of inosine into 5'-inosine acid by Pseudomonas trifolii cells was studied. The synthesis of 5'-inosine acid can be performed by both live intact and dry cells. The effectiveness of inosine phosphorylation depends on the ratio of the inosine and phosphate donor concentrations and the amount of cells. The temperature and pH effect on activity of nucleoside phosphotransferase, phosphomonoesterase and 5'-nucleotidase has been studied. The influence of surface active substances and metal ions on the synthesis of 5'-inosine acid has been investigated. Optimal conditions for the inosine transformation by the above culture have been established.
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[Effect of pH on the enzymic activity of the fungi Trichothecium roseum and Aspergillus niger hydrolyzing nonstarch polysaccharides]. The purpose of the study was to determine optimal pH values for the enzymic activity of the fungi Trichothecium roseum and Aspergillus niger hydrolyzing nonstarch polysaccharides of barley and disrupting cell walls (cytolysis) of grain, the so called cytolytic enzymes. The effect of the acidity of the medium on the stability of these enzymes was also investigated. In this connection total cytolytic activity (i. e. total activity of the enzymes hydrolyzing nonstarch polysaccharides of cell walls of barley) and hemicellulase activity of the fungi at different pH values were measured. The activity of these enzymes in aqueous extracts from the fungal culture at optimal pH after preincubation was determined at different acidity levels. The optimum of the hemicellulase activity of both fungi was at 4.6, the optimum of the total cytolytic activity of Tr. roseum at pH 5.6 and of Asp. niger at pH 3.0. The enzymes of the fungus Asp. niger showed a far higher acid stability than those of Tr. roseum.
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[Study of the yeast dissolving enzymic complex by isoelectric focusing]. By isoelectric focussing the lyzing complex produced by Actinomyces griseinus-11 has been fractionated. The sole neutral protease which is active at pH 7.0 does not participate in lysis induced by other enzymes. The lyzing activity of this complex is associated with the carbohydrase enzymes that are at least three in number. Various carbohydrases may exert a synergistic effect upon their combined action on the protein-vitamin concentrate.
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[Isolation and basic properties of thiamine pyrophosphokinase from brewing yeast]. Thiamine pyrophosphokinase (EC 2.7.7.2) isolated from dry brewing yeast has been purified 20-fold with a 70% yield. Certain properties of the enzyme have been determined: pH and temperature optima, donor and acceptor concentrations, and relationship between the rate of cocarboxylase biosynthesis and the incubation time and the enzyme quantity. The effects of concentrations of bivalent metal ions Co2+, Mg2+ and Mn2+ on the rate of the enzymic reaction has been studied. A change in the pH optimum as a function of the nature of the ion-activator has been investigated. It has been shown that neopyrithiamin is a competitive inhibitor and oxythiamin inhibits the enzymic reaction insignificantly. Thiamine phosphate cannot be transformed into thiamine diphosphate by the purified enzyme.
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[Trypsin interaction with sodium alginate]. The formation of an insoluble product of the interaction of trypsin and sodium alginate at pH 3-9 was studied. The optic density of the system was in an extreme relation to the composition. The insoluble phase was enriched in trypsin. The dispersion phase formed as a result of the electrostatic interaction between alginate macroanion and trypsin macrocation. The interaction brought about the formation of ATn, where n=90-900 (with an accuracy of the term Mw/Mn of alginate). The relation between the composition of the complex and pH was nonmonotonous.
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[Study of properties of heat activated enzyme preparations]. The paper describes studies of heat treatment of the preparations amylorizin G10x, amylosubtilin G10x and glucoendomycopsin G15 and its effect on the relationshop between the rate of the enzymic reaction and the preparation concentration, optimal pH and temperatures, and the EDTA influence on the activity of heated preparations. After heating the preparations showed an elevation of optimal temperatures of their action and a shift of optimal pH to the alkaline region. Primary heating of the preparations resulted in an increase of the rate of the enzymic reaction. Heated preparations were less susceptible to the effect of the inhibitor that nonheated ones.
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[Effect of pH on the properties of the chemostatic culture of Canida utilis]. The effect of hydrogen and hydroxyl ions on the physiological features of the yeast C. utilis VKMU-1668 was studied. High acidity inhibited yeast growth and uncoupled pathways of the energy and constructive metabolism: normal respiration was disturbed and the electron transport chain was damaged in the site of cytochromes and not flavins. Hydroxyl ions also inhibited yeast growth and uncoupled pathways of the energy and constructive metabolism: oxygen uptake and the content of flavin adenone dinucleotide increased, dehydrogenase activity upon the use of glycerol decreased significantly, and the absolute amount of all cytochromes declined slightly. The chemical composition of cellular polymers at all pH values tested was stable enough. The amount of major metabolites--volatile oils and ketoacids--was insignificant.
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[Activation of L-amino acids by aminoacyl-tRNA-synthetases from yeast Candida utilis IBPM-405]. The procedure for isolating aminoacyl-tRNA-synthetases from yeast Candida utilis IBPM-405 was developed. The rate of activation of L-amino acids in the formation of hydroxamates was different. Aspartic acid, asparagine, glutamic acid, tryptophane, phenyl alanine and methionine underwent the highest activation. The activation of alanine, arginine, hydroxyproline, serine and isoleucine was insignificant. Using aspartic acid, it was shown that the hydroxamate formation was ATP-stimulated and that the amount of hydroxamate increased with a rise of the protein concentration in the mixture to 9-10 mg/ml. The hydroxamate formation was inhibited by p-chloromercury-benzoate and heavy metal ions. Yeast aminoacyl-tRNA-synthetases showed L-aspartic and L-glutamic activities that were independent from Mg++ ions and ATP.
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[Some properties of proteases of the mould Mucor pusillus-917]. The proteolytic preparation of Mucor pusillus-917 has been obtained. The preparation produces an effective hydrolytic influence on milk casein. Thermal and acid inactivation of the proteolytic complex is the reaction of the first order. The activity-pH curves, calculations of ionization heat, inactivation of proteases by photooxidation and monoiodoacetic acid suggest that imidazole, carboxyl and sulphydryl groups of proteases are involved in the clotting and hydrolysis of milk.
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[Properties of immobilized trypsin and alpha-chymotrypsin and their use for purification of proteinase inhibitors from potatoes]. Immobilized trypsin and alpha-chymotrypsin were obtained as a result of the enzyme attachment to bromo-cyanogen activated cepharose. Proteolytic activity (substrate--casein) of immobilized trypsin and alpha-chymotrypsin was 18.7 and 9%, respectively and their esterase activity with methyl ester benzoyl-L-arginine (trypsin) and ethyl ester acetyl-L-tyrosine (alpha-chymotrypsin) was 75 and 20% of that of soluble enzymes. Immobilized enzymes were used to purify proteinase inhibitors from potatoes by affine chromatography. Specific activity of trypsin and chymotrypsin inhibitors was increased 10 and 6 times, respectively. By isoelectric focussing it was shown that the purified preparation of chymotrypsin inhibitors consisted of two acid proteins and one alkaline protein, the latter being in predominance. The purified preparation of trypsin inhibitors contained equal amounts of proteins with the isoelectric point at pH 7.1 and 8.9 and a low quantity of the component with the isoelectric point at pH 5.7.
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[Effect of the composition of the nutrient medium on the synthesis of acid-fast alpha-amylase by different strains of Aspergillus]. The capacity of 86 strains of the Aspergillus fungus to synthesize acid stable alpha-amylase was examined. The strains of Asp. niger showing a high capacity of synthesizing the enzyme were isolated. Repeated cultivation of the selected cultures on the Minoda agar medium led to a 200% increase in the enzyme activity in the submerged culture. Addition of sodium nitrate to the Minoda medium during submerged cultivation allowed a 3-fold increase of the synthesis of acid stable alpha-amylase.
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[Regulation of glutamine synthetase of the fodder yeast Candida tropicalis by ammonium ions]. The effect of ammonium on glutamine synthetase of fodder yeast Candida tropicalis was studied. Ammonium ions were found to repress the synthesis of glutamine synthetase of fodder yeast and to inhibit the enzyme in the cells. The substitution of glutamic acid for ammonium in the nutrient medium brought about depression of glutamine synthetase.
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[Rhizopus microsporus strain UzLT-I--a thermotolerant producer of lipase]. The properties of the thermotolerant fungus Rhizopus microsporus strain UzLT-1--producer of lipolyptic enzymes are described. Optimal cultivation conditions--40 degrees, C, pH 4.5--Are determined. The lipolytic activity of the culture on the medium consisting of corn extract (2%), cotton-seed oil (1%) and water is 850 ml 0.1 n KOH per 100 ml culture liquid. The enzymic preparations of lipase have been precipitated by isopropanol and ammonium sulphate. The preparation precipitated by isopropanol shows its macimum activity at pH 4.2 and 7.8 and a temperature of 40--50 degrees C.
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[Effect of different pH values on the activity and quaternary structure of asparaginase in Escherichia coli extracts]. The effect of low pH values on the activity and stability of the quaternary structure of asparaginase from Escherichia coli was investigated at early stages of purification of the enzyme. Acidification of the E. coli extract was most effective before the biomass separation. This procedure helped to separate biomass together with coagulated ballast proteins and not to reduce the activity. Upon storage of the acidified solution at 5 degrees C reversible dissociation of the tetrametric structure into dimers and monomers occurred. Stability of L-asparaginase in the storage of acetone powders and during extraction was studied. It is suggested that asparaginase in bacterial cells in unlikely to have the quaternary structure which normally occurs in the solution at neutral pH.
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[Preparation of proteolytic enzymes from the thermophilous actinomycete Actinomyces thermovulgaris str. T-54]. A preparation with a high proteolytic activity has been obtained by acetone precipitation from the culture liquid filtrate of the thermophilous actinomycete Actinomyces thermovulgaris str. T-54. The proteolytic (caseinolytic), fibrinolytic and thrombolytic activity of the preparation is comparable with that of trypsin and far superior to that of fibrinolysine. The preparation is stable at pH 5.0=9.5 and inactivated in the acid zone. The study of pH dependent proteolytic activity has shown acid, neutral and alkaline proteases in the preparation. It is relatively thermostable, and is completely inactivated for 10 min at 90 degrees. It is suggested that the preparation contains four enzymes or four enzymic groups that are different in their temperature sensitivity.
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[Analysis of acid proteinases from Aspergillus terricola]. The specific action and composition of the functional groups of active centres of three fractions of acid proteinases from Aspergillus terricola have been studied. With respect to the hydrolysis rates of acetyl-L-phenylalanyl-L-tyrosine and carbobenzoxy-D, L-glycyl-phenylalanine by the three fractions it is suggested that the interaction of acid proteinases with the substrate involves hydrophobic forces. It has been shown that the above fractions are no metal enzymes. By means of the diazocarbonyl inhibitor an occurrence of a catalytically active carboxy group has been found in the active centre of proteinases. The proteinase inhibition by Fe3+ in the presence of citric acid is an indirect evidence of the existence of several carboxy groups and, possibly, of a hydroxy group in the active centre of acid proteinases from Aspergillus terricola.
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[Preparation and properties of beta-galactosidase linked covalently with KM-cellulose]. Fungal beta-galactosidase was immobilized by covalent binding with KM-cellulose. The resultant preparation contained 3 mg protein per 1 g carrier; its specific activity was 65% of the initial one. As a result of immobilization pH optimum remained unchanged whereas the temperature optimum decreased from 65 degrees to 50 degrees. The seemings Km of the immobilized enzyme varied insignificantly as compared with Km of the soluble enzyme.
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Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase. We have examined three mutants of Klebsiella aerogenes whose genetic lesions (glnB, glnD, and glnE) are in loci unlinked to the structural gene for glutamine sythetase (glnA) and in which the control of both the level and state of adenylylation of glutamine synthetase is altered. Each mutation alters a different component of the adenylylation system of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. Inability of the cell to deadenylylate glutamine synthetase (glnB and glnD) greatly decreases its production, while inability to adenylylate glutamine sythetase (glnE) results in its constitutively high production. These results together with our previous results indicate that adenylylated glutamine synthetase inhibits the transcription of glnA.
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[Thermo- and pH-stability of soluble and silichrome-80 immobilized proteinase from Bacillus subtilis]. The stability to heating and pH changing of two proteinases of Bacillus subtilis was studied. The preparations were: protosubtilin G10x with an activity of 21500 units/g and proteinase immobilized on silochrome C-80 with an activity of 3880 units/g. For protosubtilin G10x the pH optimum was 7.0-7.2. For the immobilized preparation 96-100% activity was found at pH 5.5-10.0. The difference between the two proteinase preparations was more distinct with respect to the temperature minimum. The initial preparation-protosubtilin G10x was more stable to low temperature and showed maximum activity at 40 degrees; the immobilized preparation was less active at low temperatures and showed maximum activity at 60-70 degrees. Protosubtilin G10x was more sensitive to pH changes, especially in the acid zone (pH 4.5). It was significantly activated at 30-40 degrees and was unstable at 50-60 degrees. The immobilized preparation was activated at 50- 60 degrees and was insensitive to pH changes in the range of 4.5 to 9.2 and temperature changes from 10 degrees to 40 degrees for 1.5-2.0 hours.
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[Conditions for splitting protodioscine--the main glycoside from Tribulus terrestris L. by the enzymatic preparation from Aspergillus niger BKMt-33]. The conditions for splitting protodioscine--the main steroid saponine isolated from Tribulus terrestris L. by the enzymic preparation of Aspergillus niger str. BKMt-33 were investigated. The optimal conditions were found to be as follows: pH 4-5, temperature 30-37 degrees (the substrate concentration--5 mg%, concentration of the enzymic preparation--1%). Under these conditions the enzymolysis continued 24 hours. Mg+2 and K+ ions accelerated the reaction twice. As a result of the enzymic hydrolysis dioscine and trilline were obtained. This indicates beta-glucosidase and alpha-rhamnosidase activities of the enzymic complex isolated from Aspergillus niger str. BKMt-33.
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Neuronal properties of hybrid neuroblastoma X sympathetic ganglion cells. Clonal mouse neuroblastoma cells without tyrosine 3-monooxygenase [EC 1.14.16.2; tyrosine hydroxylase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating)] activity were fused with normal cells from embryonic mouse sympathetic ganglia. One of the 37 hybrid cell lines obtained possesses high tyrosine 3-monooxygenase activity and synthesizes dopamine. These cells also have excitable membranes and generate action potentials in response to electrical stimuli. Thus hybrid cells, generated by fusion of neuroblastoma cells with normal cells from the nervous system, can acquire neural properties not found with the parental neuroblastoma cells.
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Regulation of ferredoxin-catalyzed photosynthetic phosphorylations. Under aerobic conditions that are likely to prevail in chloroplasts in vivo, the optimal concentration of ferredoxin for cyclic photophosphorylation was found to be equal to that required for NADP reduction and about one-tenth of that needed for cyclic photophosphorylation under anaerobic conditions. In the presence of ferredoxin and NADP, cyclic photophosphorylation operated concurrently with noncyclic photophosphorylation, producing an ATP: NADPH ratio of about 1.5. The effective operation of ferredoxin-catalyzed cyclic photophosphorylation by itself required a curtailment of the electron flow from water which was accomplished experimentally by the use of either an inhibitor or far-red monochromatic light. An unexpected discovery was that the operation of cyclic photophosphorylation by itself was also regulated by a back reaction of NADPH and ferredoxin with two components of chloroplast membranes, component C550 and cytochrome b559. The significance of these findings to photosynthesis in vivo is discussed.
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High resolution 31P nuclear magnetic resonance studies of intact yeast cells. High resolution 31P nuclear magnetic resonance (NMR) spectra at 145.7 MHZ are presented for intact yeast cells. Several peaks are resolved and assigned. They include the middle phosphate peaks from long chain or cyclic polyphosphates. Our results are consistent with the suggestion that these polyphosphates act as a phosphate store in the cell. We have also been able to measure cytoplasmic pH using the orthophosphate peak inside the cell, as compared with outside the cell. The results show that yeast cells maintain their cytoplasmic pH around 6.3. This value is considerably higher than the acidic extracellular pH at which they normally live. These preliminary results indicate that 31P NMR at 145.7 MHZ can be a rapid, informative, and non-invasive method for probing biochemical events within living cells.
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Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture. Stimulation with phytohemagglutinin of the leukocytes from six of the seven known individuals with UDP-galactose 4-epimerase (= UDP-glucose 4-epimerase; EC 5.1.3.2) deficiency consistently resulted in the appearance of epimerase activity in the cultured cells. A long-term lymphoblast culture derived from one proband also contained an active epimerase enzyme. A comparison of the properties of this enzyme with those of epimerase produced by control lymphoblast lines revealed comparable Km values for UDP-galactose and NAD and identical behavior on polyacrylamide electrophoresis. However, a difference in the NAD requirement for heat stability at 40 degree provided some evidence for a structural defect in this enzyme. Possible explanations for the appearance of UDP-galactose 4-epimerase activity in stimulated lymphocytes include an increased rate of synthesis of a mutant enzyme and a derepression of an epimerase locus during lymphocyte transformation.
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beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity. beta-Bungarotoxin, a pre-synaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, has been shown to modify release of neurotransmitter at the neuromuscular junction. In this communication, we demonstrate that beta-bungarotoxin is a potent phospholipase A2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4), comparable in activity with purified phospholipase enzymes from Naja naja and Vipera russellii. The phospholipase activity of beta-bungarotoxin requires calcium and is stimulated by deoxycholate. When strontium replaces calcium, no phospholipase activity is detected. Since neuromuscular transmission is not blocked when calcium is replaced by strontium, it was possible to examine the effects of the toxin on neuromuscular transmission in the presence of strontium. Under these conditions, when the phospholipase activity should be inhibited, the toxin has little or no effect on neuromuscular transmission. If beta-bungarotoxin owes its toxicity in part to its enzymatic activity, then it must be placed in a different class from those toxins which produce their effect by binding passively to an appropriate receptor.
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Isolation and characterization of an unsaturated fatty acid-requiring mutant of cultured mammalian cells. An unsaturated fatty acid-requiring mutant derived from Chinese hamster ovary (CHO) cells has been isolated and characterized. This mutant grows normally when oleate or other unsaturated fatty acids are supplemented in the growth medium. Unlike the wild-type CHO cells, growth stops when medium is deprived of unsaturated fatty acid. Whole cell pulse experiments with [14C]acetate or [14C]stearate indicate that the mutant is defective in unsaturated fatty acid synthesis. Enzyme assays in vitro show that the enzymatic defect of the mutant is localized to the microsomal stearoyl-CoA desaturase.
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DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum. The method of DNA alkaline elution was applied to a study of the formation and resealing of DNA single-strand breaks after irradiation of human fibroblasts with ultraviolet light (UV). The general features of the results were consistent with current concepts of DNA excision repair, in that breaks appeared rapidly after UV, and resealed slowly in normal fibroblasts, whereas breaks did not appear in those cells of patients with xeroderma pigmentosum (XP) that are known to have defects in DNA repair synthesis. The appearance of breaks required a short post-UV incubation, consistent with the expected action of an endonuclease. Cells of the variant form of XP characterized by normal DNA repair synthesis exhibited normal production of breaks after UV, but were slower than normal cells in resealing these breaks. This difference was enhanced by caffeine. A model is proposed to relate this finding with a previously described defect in post-replication repair in these XP variant cells. DNA crosslinking appears to cause an underestimate in the measurement of DNA breakage after UV.
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Modifications of purified glucose-6-phosphate dehydrogenase and other enzymes by a factor of low molecular weight abundant in some leukemic cells. Highly purified platelet glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) can be modified in its isoelectric point and its molecular specific activity by extracts of some leukemic granulocytes. The "G6PD modifying factors" are relatively small molecules (molecular weight slightly under 5000), thermostable, dialyzable, and ultrafilterable. These molecules are destroyed by various endo- and exopeptidases and by serine enzymes present in crude extracts of leukocytes and commercial preparations of ribonuclease. The alterations of platelet G6PD due to the "G6PD modifying factors" are stable and not reversible by dialysis or further chromatography. The leukemic extracts which are able to modify G6PD also can modify the electrophoretic mobility and (or) the enzymatic activity of purified leukocyte pyruvate kinase, 6-phosphogluconate dehydrogenase, and glucosephosphate isomerase. The chemical nature of such modifications and their relationships with post-translational modifications which occur in leukemic or normal cells are discussed.
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Copper-induced activation of aortic lysyl oxidase in vivo. Raising day-old chicks on diets lacking copper severely depressed the activity of lysyl oxidase, a copper metalloenzyme in connective tissue. Administration of CuSO4 either through the diet or through intraperitoneal injections restored the lysyl oxidase activity in aortic tissue. Two hours after the chicks received CuSO4 (1 mg/kg) the activity of lysyl oxidase rose rapidly to attain, within 4-6 hr, a new steady-state level which was five to 20 times higher than the basal (saline-injected) activity. Twenty hours after copper administration, activity was still higher, in some experiments double that achieved at 6 hr. Very low amounts of cycloheximide injected intraperitoneally 45 min before and 3 hr after copper suppressed the activation response by two-thirds. Cycloheximide given 2 or 4 hr after the copper was only one-half as effective. Actinomycin D caused only a 10-15% inhibition of the copper-induced activation. The data suggest that copper is a key regulator of lysyl oxidase activity in aorta and may in fact be a major determinant of the steady-state levels of the enzyme in that tissue.
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Evidence that acyl coenzyme A synthetase activity is required for repression of yeast acetyl coenzyme A carboxylase by exogenous fatty acids. The cellular content of acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] in Saccharomyces cerevisiae is reduced by the addition of long-chain fatty acids to the culture medium. Mutant strains of S. cerevisiae defective in acyl-CoA synthetase [acid:CoA ligase (AMP-forming), EC 6.2.1.3] were isolated and used to determine whether fatty acid itself or a metabolite of fatty acid is more directly responsible for the repression of acetyl-CoA carboxylase. Cells of the mutant strains were capable of incorporating fatty acid to an extent comparable to that observed with the wild-type strain, but they accumulated markedly more of the incorporated fatty acid in the nonesterified form than did the wild-type cells. The level of acetyl-CoA carboxylase activity in the mutants, in contrast to that in the wild-type strain, was hardly affected by the addition of fatty acids to the medium. These results indicate that the activation of exogenous fatty acid is required for the repression of acetyl-CoA carboxylase, supporting the view that the repressive effect is mediated by some compound metabolically derived from fatty acid.
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Glutathione and gamma-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa. Villus tip cells and crypt cells of rat jejunal mucosa were separated by the planning procedure of Imondi et al. and were studied with respect to their activities of the enzymes of the gamma-glutamyl cycle and glutathione content. The villus tip cells exhibit much higher gamma-glutamyl transpeptidase activities than do the crypt cells: thus, gamma-glutamyl trnaspeptidase appears to be a villus-specific enzyme. gamma-Glutamyl cyclotransferase and the enzymes required for glutathione synthesis are not specifically localized to either the crypt or villus tip cells but are present in both. The crypt cells have a high concentration of glutathione (4-5 mM) comparable to the levels found in liver and kidney; in contrast, the villus tip cells have much lower concentrations. On fasting, the glutathione concentration decreased markedly in both villus tip and crypt cells; feeding of protein, but not of sucrose, led to increased glutathione concentrations. The migration of cells from the undifferentiated crypt cell region to the villus tip is associated with structural and biochemical changes that equip the cell for its mature functional activities, which include transport. The present findings indicate that such cellular differentiation and migration is associated with a marked increase in gamma-glutamyl transpeptidase activity and in the utilization of glutathione.
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Maturation of neuroblastoma cells in the presence of dimethylsulfoxide. Addition of dimethylsulfoxide at concentrations of 1% and 2% (vol/vol) to cells of mouse neuroblastoma clone NIE-115 in the confluent phase of growth resulted in the production of morphologically differentiated cultures with extensive process formation. Cell maintained in 2% dimethylsulfoxide remained in a stable nondividing condition for periods of up to 4 weeks. A high degree of electrical excitability was found in these cells, but there was no clear correlation of this property with the level of induction of either acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2]. In addition, intracellular levels of cyclic 3':5'-AMP were not elevated in fully morphologically and electrically differentiated cells. While cell division was markedly inhibited by 2% or higher concentrations of dimethylsulfoxide, at 1% growth continued at a somewhat slowed rate and such cultures exhibited enhanced process formation and electrical activity for a relatively short period. High concentrations (3% or 4%) of dimethylsulfoxide totally suppressed process formation and did not result in increased excitability, but cells maintained high resting potentials. The results suggest that the development of the excitable membrane in neuroblastoma cells may be expressed independently of neurospecific enzyme induction, and does not require a sustained elevation of cyclic 3':5'-AMP levels.
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Properties of a toxin from the sea anemone Stoichacis helianthus, including specific binding to sphingomyelin. Stoichactis helianthus toxin, a protein derived presumably from the nematocysts, was purified to homogeneity. It has a molecular weight of about 16,000, an isoelectric pH of 9.8, and it contains approximately 3.7% carbohydrate. It is powerfully hemolytic for erythrocytes derived from a variety of animal species, those of the cat being the most sensitive and those of the guinea pig the most resistant. The toxin is lytic also for rabbit blood platelets, and it destroys cultured fibroblasts but is inactive for several kinds of bacterial protoplasts and spheroplasts. The hemolytic activity is specifically inhibited by sphingomyelin, and it is proposed that this phospholipid is the constituent of the membrane which functions as receptor for the toxin. Supporting evidence includes the findings that enzymes known to destroy sphingomyelin (a) prevent erythrocyte membranes from inhibiting hemolysis, and (b) render erythrocytes resistant to lysis by the toxin. The mechanism underlying hemolysis may involve translocation of membrane sphingomyelin by virtue of a specific affinity of the coelenterate protein for this phospholipid.
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Mechanistic studies of glutamine synthetase from Escherichia coli: kinetic evidence for two reaction intermediates in biosynthetic reaction. Fast reaction techniques were used to study the kinetics of protein fluorescence intensity changes that are associated with the reactions of unadenylylated Escherichia coli glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] with its substrates. It was established that the synthesis of glutamine occurs by a stepwise mechanism. During the catalytic process two fluorometrically distinct intermediates were observed. Both forward and reverse rate constants which lead to the formation and consumption of these intermediates were evaluated. The catalytic rate constant, kc, which was calculated from these rate constants agrees well with the values of kc which were determined by direct measurement of the overall biosynthetic activities by means of stopped-flow technique or the steady-state assay method.
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Molecular mechanism of inhibition of firefly luminescence by local anesthetics. The kinetics of the action of local anesthetics upon firefly luciferin and luciferase systems is presented. Clinical concentrations of local anesthetics inhibited this ATP-induced luminescence in a dose-dependent manner. From the effects of temperature and pH upon the inhibitory action of the local anesthetics, it is concluded that hydrophobic ligand-enzyme interaction is the predominant cause of the inhibition, but hydrophilic interaction also contributes to the inhibition to a lesser degree. A molecular theory of anesthesia is outlined which postulates that release of electrostricted water molecules from the hydrophilic parts of the enzyme due to the protein conformational changes induced by anesthetics is the cause of the decreased luminescence. A similar mechanism is expected to occur at the cell membrane, which probably dehydrates the sodium channel and suppresses the conductance of this ion across the membrane. These events lead to a volume expansion of the total system, and the system becomes reactive to a pressure which reverses the anesthesia by shifting the equilibrium to the nonanesthetized original volume. The pressure antagonism of anesthesia can be explained by this overall volume expansion and not by a mere swelling of the cell membrane.
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Cyclic nucleotide metabolism in compensatory renal hypertrophy and neonatal kidney growth. Cyclic nucleotide metabolism was investigated in growing kidneys of rats during compensatory hypertrophy and during neonatal development. After unilateral nephrectomy a mild and short-lasting decrease in cyclic 3':5" adenosine monophosphate (cAMP) was observed in the hypertrophying kidney. In contrast, cyclic 3':5' guanosine monophosphate (cGMP) showed a sharp decline to 20% of control at 15 min and a rapid rise to 200-300% above base-line at 1-72 hr. The alterations in renal tissue levels of cGMP were associated with parallel changes in the soluble, 100,000 X g supernatant guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing): EC 4.6.1.2]. No change was observed in total cGMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17). In the rapidly growing kidney of newborn rats cAMP levels were 983 +/- 65 and 833 +/- 42 pmol/g of kidney at 4 and 7 days after birth, and increased to adult levels (1518 +/- 57 pmol/g) at 21 days whereas cGMP levels were 59.8 +/- 6.8 and 92.5 +/- 13.9 pmol/g at 4 and 7 days and decreased to adult levels (36 +/- 1.5) at 21 days. The results indicate that compensatory renal hypertrophy and neonatal kidney growth are associated with changes in cAMP and cGMP metabolism.
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Effects of denaturants on the sweet-tasting protein monellin. Effects of the denaturants urea and guanidine-HCl on the sweet-tasting protein monellin have been studied. The pH at which monellin is initially treated with denaturant is an important factor in retention of sweetness, but the pH maintained during subsequent removal of denaturant by dialysis has no effect on activity. Recovery of sweetness of denaturant-treated monellin is favored when denaturation occurs at acid pH. Monellin treated with either 6 M guanidine-HCl or 8 M urea at acid pH retains all of its sweetness following removal of denaturant, but urea treatment at neutral pH leads to some irreversible loss of sweetness. Monellin precipitates from solution under some conditions during removal of denaturant by dialysis, and the precipitated protein is no longer sweet. Precipitation is least under acid conditions. Aggregated protein was demonstrated by gel filtration chromatography. The single sulfhydryl group of monellin was not demonstrable in the precipitated protein, having apparently become oxidized during denaturation and formation of the aggregated protein. The data support the hypothesis that the tertiary structure is important in the ability of monellin to elicit a sweet sensation.
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Thyroxine deiodination associated with NADPH-dependent lipid peroxidation in a submicrosomal system. A lipoprotein present in trypsin-treated microsomes can be oxidized with formation of malondialdehyde in a system which contains NADPH, ferric ion-ADP complex, NADPH-cytochrome c reductase and a factor. This factor, a mixture of peptides, can be isolated from hepatic microsomes by trypsin digestion and successive gel filtration through Sephadex G-100 and G-25 columns. Lipid peroxidation in this system catalyzes the deiodination of thyroxine, as does NADPH-dependent lipid peroxidation in fresh hepatic microsomes. Thyroxine inhibits lipid peroxidation as it is deiodinated in this system.
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Effect of environmental pH on adenovirus-associated virus. The influence of environmental pH on AAV was studied in infectious virus titrations, induction of CF antigens production of infectious virus, induction of immunofluorescent stainable antigen, and aggregation of the viral particles. The pH of the medium was found to influence the titer of virus stocks in that less virus was registered at acid pH's, giving differences of up to 105 TCID50 in HEK and HEp-2 cells. Less infectious virus was produced in KB cells, and decreased amounts of CF antigen appeared at acid pH's. However, increased levels of detectable intracellular FA antigen appeared at acid pH's. Electron microscopic examination of AAV particles negatively stained at various pH's showed increasingly large aggregates of particles as the pH was lowered. Under the acid conditions studied, the adenovirus helper and cell activities were only slightly suppressed, with the greatest effect due to aggregation of the virus particles.
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The effect of dexamethasone on urinary acidification. Although it is well recognized that mineralocorticoids enhance renal acid excretion, the effect of glucocorticoids on renal acidification is unclear. Oral administration of dexamethasone to six healthy volunteers for 1 week at a daily dose of 4.5 mg was associated with mild respiratory alkalosis and a small but statistically significant increase in baseline urine pH. However, neither the ability to lower urine pH nor to excrete titratable acid and ammonium after NH4Cl acid-loading was altered. Administration of a single intravenous dose of dexamethasone sodium phosphate (7.5 mg) was associated with a significant rise in urine pH and potassium excretion and decreased titratable acid, ammonium , and phosphorus excretion in the absence of changes in blood acid-base status, creatinine clearance, or urine flow.
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A rapid method for measuring guanylate cyclase activity in mammary tissue. A simple and rapid method for measuring guanylate cyclase activity in broken cell preparations of biological tissues is described. This method employs the rate of conversion of [32P]GTP to [32P]cyclic-GMP. The product of this reaction is isolated by ion-exchange chromatography and by a ZnSO4-Ba(OH)2 precipitation at pH 5.7. Using this method, about 30-50 samples can be assayed for guanylate cyclase activity during a 5-6 hr period. The characteristics of this enzyme in the mammary gland were found to be similar to those described for other tissues using different methods for measuring guanylate cyclase activity.
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Guanylate cyclase and cyclic-GMP phosphodiesterase activities in mammary glands of mice during pregnancy and lactation. Guanylate cyclase and cyclic-GMP phosphodiesterase activities were measured in homogenates of mammary glands from virgin, pregnant, and lactating mice. Guanylate cyclase activities increased 35% in mammary tissues during pregnancy, and a further 40% increase was observed during lactation. Cyclic-GMP phosphodiesterase activity also increased during pregnancy but activities were not different in glands from lactating mice vs glands from pregnant mice. These results are discussed with regard to a possible role of cyclic-GMP in regulating lactational processes.
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Cyclic-AMP and pancreatic bicarbonate secretion in response to secretin in dogs. In anesthetized dogs given secretin intravenously in doses doubling every 60 min and ranging from 0.5 to 8 units per kg body weight per hr, cyclic-AMP levels in pancreatic tissue rose continuously, whereas DNA concentrations were slightly decreased. Bicarbonate concentrations and bicarbonate outputs, cyclic-AMP tissue concentrations and bicarbonate outputs, as well as cyclic-AMP tissue concentrations and juice outputs, were significantly correlated. In conscious pancreatic fistula dogs, there was also a significant correlation between cyclic-AMP and bicarbonate concentrations and outputs in the pancreatic juice after stimulation by exogenous secretin. Accordingly, enhanced release of endogenous secretin achieved by intraduodenal acidification led to a dose-dependent increase in bicarbonate and cyclic-AMP outputs in both conscious and anesthetized dogs. Phosphodiesterase inhibitors (aminophylline, caffeine, and papaverine) given alone to the conscious dogs did not initiate pancreatic bicarbonate secretion, but they potentiated bicarbonate responses to exogenous secretin. These data suggest that cyclic-AMP plays a part in secretin-stimulated pancreatic secretion.
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Effects in the rat of intradermal injection of purified proteinases from streptococcus and Serratia marcescens. Purified streptococcal proteinase and Serratia proteinase are potent permeability factors in rat skin and initiate histopathological evidence of an acute inflammatory response. These effects appear to be largely independent of terminal components of complement, histamine, and polymorphonuclear leukocytes.
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Effect of recurrent stress on postnatal increase of tyrosine hydroxylase. Tyrosine hydroxylase is present at birth and reaches adult levels in the hypothalamus usually during the second month. Recurrent stimulation of intrahypothalamic noradrenergic structures shortened this period of maturation in a statistically significant manner.
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Comparison of beta-adrenoceptor blocking properties of sotalol, oxprenolol, propranolol and pindolol on rabbit intestinal smooth muscle. Adrenergic agonists produced a characteristic and definite decrease in the amplitude of spontaneous contractions and tone of the isolated rabbit jejunum. Effect of phenylephrine was abolished either by phenoxybenzamine or phentolamine. Relaxation induced by epinephrine and by norepinephrine was inhibited after combined treatment with phentolamine and propranolol. Phentolamine alone diminished the response to epinephrine and to norepinephrine, but the diminution for epinephrine was greater, indicating that epinephrine has a greater affinity for alpha- than for beta-receptors in the rabbit jejunum. Stimulation of the beta-receptors by isoproterenol was inhibited by propranolol, oxprenolol, sotalol and pindolol, but the block was incomplete. The activity of these four beta-blockers in preventing the inhibitory response to isoproterenol was as follows: inidolol greater than or equal to oxprenolol greater than propranolol greater than sotalol. This demonstrates the fact that not all beta-adrenergic blocking agents possess an identical pharmacologic spectrum of activity. Also it can be suggested that the beta-receptors of jejunum differ in specificity from those of other organs.
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Effect of propranolol on antinociceptive and withdrawal characteristics of morphine. Propranolol at a dose (10 mg/kg) which did not alter tail-flick latency by itself, did not alter the ED50 of morphine when given 10 min prior to the narcotic. Propranolol at doses of 10 and 25 mg/kg given 10 min prior to naloxone challenge did not significantly alter the frequency of naloxone induced jumping 72 hr after morphine pellet implantation. The ED50 of naloxone in morphine pelleted mice was not altered by treatment with propranolol at 0, 24, and 48 hr after pellet implantation. Naloxone caused hyperactivity in mice when administered 72 hr after morphine pellet implantation. An injection of 25 mg/kg propranolol 10 min prior to naloxone did not block this hyperactivity. In addition, administration of 10 mg/kg of propranolol every 8 hr to rats during withdrawal from morphine failed to alleviate the withdrawal syndrome as evidenced by changes in either body weight or water intake. These data suggest that the beta-adrenergic blocking agent, propranolol, does not alter the antinociceptive activity or lessen the withdrawal syndrome of morphine in rodents.
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The antinicotinic effects of drugs with clinically useful sedative-antianxiety properties. Mice were given a drug per os and 2 h later were challenged with an intravenous LD95 of nicotine. Amitriptyline, imipramine, doxepin, meprobamate, chlordiazepoxide, diazepam, trifluoroperazine, haloperidol, thioridazine, chlorpromazine, phenobarbital, propranolol and diphenylhydantoin were all active in protecting mice from extensor convulsions and lethality. Iproniazid, tranylcypromine, atropine, benztropine and trimethadione were inactive. There appears to be a relationship between blockage of nicotine-induced extensor convulsions and lethality in mice and sedative-antianxiety effects in man. This relationship is especially good for drugs designated antidepressant, antianxiety and antipsychotic.
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Increased acquisition of a complex appetitive task after MSH and MIF. After daily injections of melanocyte stimulating hormone (MSH), MSH-release inhibiting factor (MIF), or diluent albino rats ran a 12 choice Warden maze for a palatable food reward. Rats receiving the hormones had shorter latencies and made fewer errors than controls during learning but, unlike results with simple tasks, there were no differences during extinction. The results demonstrated that both MSH and MIF-I could facilitate the acquisition of an appetitive task which seemed of sufficient complexity to emphasize differences in performance.
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The jumping mechanism of Xenopsylla cheopis. I. Exoskeletal structures and musculature. The jumping apparatus of the flea, which includes highly modified direct and indirect flight muscles, is described: attention is drawn to the various specializations of the exoskeleton which stiffen the thorax and also provide the 'click' mechanism triggering take-off. A finger-like invagination of tall cells within the cavity of the developing pleural arch of the pharate adult secretes the resilin pad. This is illustrated with coloured photographs. It is suggested that winglessness of a Mecopteran-like ancestor pre-adapted fleas to a parasitic life-style, and that a jumping mode of progression was a primitive feature of the whole Order. Scattered throughout the Siphonaptera today are species which have secondarily lost the pleural arch and with it the power to execute large jumps. These are usually found among fleas parasitizing mammals inhabiting caves, subterranean burrows and runs, high aerial nests and snow or ice-bound habitats. Large pleural arches are associated with fleas infesting large mobile hosts.
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The jumping mechanism of Xenopsylla cheopis. II. The fine structure of the jumping muscle. The ultrastructure of the trochanteral depressor muscle of the oriental rat flea is described. It is shown to be similar to that of the tubular leg muscles of other insects except in the volume and arrangement of the sarcoplasmic reticulum. The sarcoplasmic reticulum occupies approximately 18% of the volume of the muscle fibres. It is in three configurations:a regular array of parallel tubules opposite the A-band, a collar of sacculi involved in the formation of the dyads at the edge of the A-band and a loosely woven arrangement of tubules around the I-band. This tripartite arrangement of the sarcoplasmic reticulum and its large surface area is discussed in relation to the action of the muscle as the main propulsive muscle in the jump of the flea.
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The jumping mechanism of Xenopsylla cheopis. III. Execution of the jump and activity. The flea's hind legs are the chief source of jumping power, but in species which execute large jumps, take-off is accelerated by elastic energy released from a resilin pad (homologous with the wing hinge ligaments of flying insects) situated in the pleural arch. A central click mechanism, operated by a rapid twitch of the trochanteral depressor (the starter muscle), synchronizes the separate sources of energy which power the jump. Ciné photos confirm the morphological evidence that the flea takes off from the trochanters, not the tarsi. The loss of wings, associated with lateral compression of the body and the shortening of the pleural ridge (which thus lowers the position of the pleural arch) together with modifications of the direct and indirect flight muscles, are some of the main morphological features associated with the change from a flying to a saltatorial mode of progression. The flea's take-off basically resembles that of other Panorpoid insects (Diptera, Mecoptera, etc.). The release of elastic energy from the pleural arch is a system by which the force used to move the wings of flying insects is rapidly fed back into the legs and adds power to the jump.
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Dynamic aspects of enzyme specificity. There are two aspects of enzyme specificity: recognition of the substrate by the formation of an enzyme-substrate compound and recognition of the transition state by catalysis of the reaction. Kinetic studies with inactive substrate analogues as potential competitive inhibitors, and structural studies of their compounds with enzymes, give information about the first of these specificity elements. Comparative kinetic studies with alternative substrates give information about both. There is a great deal of information from kinetic studies of dehydrogenases about the coenzyme specificities, substrate specificities and stereospecificities and mechanisms of these enzymes, particularly alcohol dehydrogenases. Recent X-ray diffraction studies of dehydrogenases have given insight into the molecular basis of some of their specificity elements. An attempt is made to correlate the available kinetic and structural data for alcohol and lactate dehydrogenases.
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Self-assembly of biological macromolecules. The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.
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Water structure and hydration. Possible structures adopted by bulk water are discussed with special reference to the possible presence of monomeric water and the detection of 'free' -OH groups. The way in which water tends to accommodate small hydrophobic molecules is considered, with particular reference to the clathrate theory and the phenomenon of 'structure making'. Cage-pairing and cage-sharing processes are described. Consideration of the way water solvates cations and anions is followed by a discussion of the way these solvated ions interact with the bulk medium. Large symmetrical alkylammonium ions probably encourage clathrate cage formation, at least at low temperatures. Particular reference is made to the use of infrared, Raman, ultraviolet, n.m.r. and e.s.r. spectroscopic techniques to the study of water and aqueous solutions.
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Drug-nucleic acid interaction: X-ray crystallographic determination of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3'-5')adenosine. The intercalative trypanosomal drug, ethidium bromide, forms a crystalline complex with the dinucleoside monophosphate, 5-iodiuridylyl(3'-5')adenosine (iodoUpA). These crystals are monoclinic, space group C2, with unit cell dimensions, a = 2.845 nm, b = 1.354 nm, c = 3.413 nm, beta = 98.6 degrees. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least squares to a residual of 0.29 on 2017 observed reflexions. The asymmetric unit contains two ethidium molecules, two iodoUpA molecules, twenty water molecules and four methanol molecules, a total of 156 atims excluding hydrogens. The two iodoUpA molecules are held together by adenine-uracil Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoUpA structure and between neighbouring iodoUpA molecules in adjoining unit cells are separated by 0.68 nm. This separation results from intercalative binding by one ethidium molecule and stacking by the other symmetry is utilized in this model drub-nucleic acid interaction, the intercalative ethidium molecule being oriented such that its phenyl and ethyl groups lie in the narrow groove of the miniature nucleic acid double helix. Solution studies have indicated a marked sequence specificity for ethidium-dinucleotide interactions and a probable structural explanation for this has been provided by this study.
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DNA-RNA hybridization. Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.
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The dynamic properties of biological membranes. Biological membranes must be viewed as highly dynamic, undergoing continuous structural fluctuations and changes in response to external perturbations. The study of liposomes by 31 P n.m.r. and fluorescence can reveal some of the motional characteristics of the different regions in a bilayer. Asymmetric lipid distribution and how this depends on the environment is also observed by n.m.r. The nature of the interaction of amine anaesthetics and of polypeptide antibodies with membranes is discussed in relation to their perturbing effect. The role of lipid mobility in modulating hormone-receptor interaction is discussed with reference to the binding of thyroid stimulating hormone.
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Recognition at cell surfaces: phytohaemagglutinin-lymphocyte interaction. Many aspects of cell behaviour are regulated by the interaction of extracellular ligands with specific receptors exposed on the cell surface. The receptors correspond to membrane proteins and expecially glycoproteins. A key event in regulation is the transmission across the surface membrane of the information resulting from receptor-ligand interaction. The activation of lymphocytes by Phaseolus vulgaris phytohaemagglutinin (PHA) provides a convenient experimental model for the study of the molecular basis of receptor-ligand interaction and the molecular consequences of interaction. The receptor mediating lymphocyte activation by PHA is probably a unique glycoprotein which is present to the extent of about 3 X 10(4) molecules/cell. The PHA-receptor complex solubilized in 1% sodium deoxycholate has a molecular size of about 3 X 10(5). The primary event in the activation process is probably an increase in the permeability of the surface membrane to Ca2+. This may be achieved by PHA cross-linking ('patching') the receptors to form a polar channel that permits an influx of Ca2+.
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Cell communication by periodic cyclic-AMP pulses. At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cycl-nucleotide phosphodiesterase. Both sites are developmentally regulated. An adequate stimulus for the chemoreceptor system in D. discoideum is the change of cAMP concentration in time, rather than concentration per se: long-term binding of cAMP causes only short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The ce,lls are, nevertheless, able to sense stationary spatial gradients and to respond to them by chemotactic orientation. The possibility is discussed that they do so by transforming spatial concentration changes into temporal ones, using extending pseudopods as sensors. The cAMP recognition system is part of a molecular network involved in the generation of spatio-temporal patterns of cellular activities. This system controls the periodic formation of chemotactic signals and their propagation from cell to cell. The phosphodiesterase limits the duration of the cAMP pulses and thus sharply separates the periods of signalling; the binding sites at the cell surface are supposed to be the chemoreceptors. The control of cellular activities via cAMP receptors can be studied with biochemical techniques with cell suspensions in which spatial inhomogeneities are suppressed by intense stirring, whereas the temporal aspect of the spatiotemporal pattern is preserved. Under these conditions it can be shown that the extracellular cAMP concentration changes periodically, and that the phase of the cellular oscillator can be shifted by external pulses of cAMP. It can also be shown that small cAMP pulses induce a high output of cAMP, which demonstrates signal amplification, a function necessary for a cellular relay system.
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Recognition of metal cations by biological systems. Recognition of metal cations by biological systems can be compared with the geochemical criteria for isomorphous replacement. Biological systems are more highly selective and much more rapid. Methods of maintaining an optimum concentration, including storage and transfer for the essential trace elements, copper and iron, used in some organisms are in part reproducible by coordination chemists while other features have not been reporduced in models. Poisoning can result from a foreign metal taking part in a reaction irreversibly so that the recognition site or molecule is not released. For major nutrients, sodium, potassium, magnesium and calcium, there are similarities to the trace metals in selective uptake but differences qualitatively and quantitatively in biological activity. Compounds selective for potassium replace all the solvation sphere with a symmetrical arrangement of oxygen atoms; those selective for sodium give an asymmetrical environment with retention of a solvent molecule. Experiments with naturally occurring antibiotics and synthetic model compounds have shown that flexibility is an important feature of selectivity and that for transfer or carrier properties there is an optimum (as opposed to a maximum) metal-ligand stability constant. Thallium is taken up instead of potassium and will activate some enzymes; it is suggested that the poisonous characteristics arise because the thallium ion may bind more strongly than potassium to part of a site and then fail to bind additional atoms as required for the biological activity. Criteria for the design of selective complexing agents are given with indications of those which might transfer more than one metal at once.
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Structure and specificity of antibody molecules. The structure of the Fab' fragment of a human myeloma protein (IgG1 (lambda) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tighly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.
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Intermolecular forces. The nature of molecular interactions is examined. Intermolecular forces are divided into long-range and short-range components; the former operate at distances where the effects of electron exchange are negligible and decrease as an inverse power of the separation. The long-range interactions may be subdividied into electrostatic, induction and dispersion contributions, where the electrostatic component is the interaction of the permanent charge distributions and the others originate in the fluctuations in the distributions. Typical magnitudes of the various contributions are given. The forces between macroscopic bodies are briefly considered, as are the effects of a medium. Some of the manifestations of molecular interactions are discussed.
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Antibody--hapten interactions in solution. This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.
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Molecule--receptor specificity. The specificity of binding between small molecules and macromolecular receptors may be studied by comparing theoretically calculated conformational potential energy surfaces of a series of chemically similar molecules which have a range of receptor binding energies and biological activities. In this way essential requirements for binding may be highlighted, including the necessity of the small molecule adopting, or passing through, conformations which are not at energy minima and not found either in the solid state or in aqueous solution. In particular the conformational demands of the adrenergic beta-receptor and histamine H1 receptor are considered.
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Enzyme substrate and inhibitor interactions. An enzyme is designed to bind most tightly to a substrate when it is in the transition state of the reaction which the enzyme catalyses. The consequent reduction of the activation energy of the reaction constitutes the catalytic mechanism. The energetic contributions of different features of the interaction can only be crudely assessed, but they are dominated by entropically driven effects. The binding site of trypsin orients the substrate so that the reacting groups are correctly placed for reaction to occur. Apart from two side chains which take part in chemical steps of the reaction, the enzyme behaves almost as a rigid body. The full binding interactions are only developed when the substrate is in an intermediate stage of the reaction. The tightly bound complexes of trypsin with protein trypsin inhibitors have proved amenable to structural analysis. Enzyme inhibitor interactions, which account for almost 80 kJ mol-1 of interaction energy, are known fairly accurately. The similarity of the two known trypsin inhibitor structures, close to the primary binding site, indicates a high specificity, even for this simple interaction. In cases where no large conformational changes occur the specificity of an enzyme should be predictable from accurate knowledge of its tertiary structure.
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The thermal transition of brain soluble proteins. Soluble rat brain proteins undergo a thermal reversible denaturation in the range of 20 degrees C -65 degrees C. The thermal transition as studied in 0.25 M sucrose solution, is associated with changes in the proteins ionization capacity by the lowering of the isoionic solution pHfrom a value of 6.95 at 20 degrees C to 6.55 at 65 degrees C. The apparent enthalpy change delta H at the transition temperature (t=50 degrees C) is about 34 Kcal, heat capacity delta Cp about 1.75 Kcal, and apparent entrophy change deltaS 100 e.u. The data suggest that the thermal transition is predominantly a two-state process. A prolonged keeping of the protein solution at the increased temperature produces a partial reversibility of thermal transitions by a lowering of 5-HT cations fixing on the protein molecule.
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