Acid-base changes in milk and blood of rats in acidosis and alkalosis. Lactating white rats (Rattus norvegicus) were subjected to metabolic and respiratory acidosis and metabolic alkalosis. Before and during the various treatments, the acid-base status of heart blood and milk was determined. Acute metabolic acidosis lowered the pH of plasma and milk; Pco(2) and bicarbonate concentrations in plasma were lowered, and in milk Pco(2) was raised and the bicarbonate concentration remained unchanged. Respiratory acidosis and acetazolamide caused a drop in blood pH and in blood and milk bicarbonate concentrations; milk pH remained unchanged, but Pco(2) was raised in both plasma and milk. Acute metabolic alkalosis raised the blood pH and milk Pco(2); plasma Pco(2) and bicarbonate concentrations in blood and milk remained unchanged. The data show that greater changes occur in acid-base parameters of blood than milk when animals are exposed to acidifying and alkalinizing stimuli.


Role of oxalacetate in lipoate effect on frog gastric mucosa. The mechanism of action of lipoate on frog gastric mucosa was investigated. Oxalacetate (OAA) reversed lipoate-inhibited QO2 and QH+ of chambered mucosas by 70 and 40%, respectively. Pyruvate or glucose produced similar effects. Neither activity was affected by OAA when added after glucose, pyruvate, decanoate, butyrate, or lipoate-propionate-inhibited mucosa. Lipoate-treated or lipoate-propionate-treated mucosa did not respond to histamine; OAA addition prior to histamine restored responsiveness. Tracer and chromatographic techniques showed that lipoate reduced and pyruvate increased OAA formation. Preincubation of mitochondrial extracts of gastric mucosa with 2 mM lipoate increased pyruvic dehydrogenase activity 110%. Pyruvic carboxylase (PC) activity was primarily in the mitochondrial fraction of the gastric mucosa. The PC preparation was shown to have an absolute requirement for CoASAc, contained biotin, was not inhibited by lipoate, and had an apparent Km approximately equal to 3.6 X 10(-4) M for pyruvate. The results suggest that OAA concentration is regulated by PC activity and is one of the factors controlling QO2 and QH+ in the frog gastric mucosa.


Sensitivity of small subcutaneous vessels to altered respiratory gases and local pH. Television microscopy was used to quantitate the responses of small arteries and veins, in the wings of unanesthetized bats, to alterations in the inspired concentrations of O2 and CO2. Mean arterial pressure, heart rate, and the diameters of small arteries (28-54 mum) and veins (50-128 mum) were measured during a 90-min protocol--30 min with an inspiratory gas mixture of 20% O2 and 80% N2 (control period); 30 min with a gas mixture containing 5% O2 (hypoxic period) or 12, 20, or 28% CO2 (hypercapnic period); and 30 min with the original control gas. The hypoxic responses were dilatation of arteries and no change in the veins in both innervated and surgically denervated wings. Hypercapnia resulted in artery dilatation in innervated wings. Hypercapnia resulted in artery dilatation in innervated wings and constriction in denervated wings. The veins constricted in both innervated and denervated wings during the hypercapnia period. In another series, topical application of Krebs solutions (pH ranging from 7.7 to 6.7) to exposed segments of small arteries and veins produced dilatation of both vessels with decreasing pH. Artery dilatation during hypoxia and vein constriction during hypercapnia involve non-neural mechanisms, while both a neural stimulus for dilatation and a non-neural stimulus for constriction are components in the response of innervated arteries to hypercapnia. The non-neural stimulus for artery and vein constriction during hypercapnia is not a local decrease in pH.


Hypothalamic and thalamic blood flow during somatic afferent stimulation in dogs. The effect of somatic afferent C fiber stimulation on regional cerebral blood flow (rCBF) and cerebral tissue available oxygen (aO2) was studied in 20 dogs under chloralose anesthesia. Mean arterial blood pressure, arterial Pco2, and pH were stabilized before and during the 3-min stimulation of the sciatic nerves (20 V, 300 ms, 15 Hz). Combined gold+platinum electrodes were chronically implanted into the ventral posterolateral nucleus of the thalamus, ventromedial nucleus of the hypothalamus, and into the white matter. Tissue aO2 and rCBF of these regions were measured polarographically, the latter by the H2-gas clearance technique. Blood flow changed from 42 +/- 2.1 to 28 +/- 1.7 ml/100 g per min (cerebrovascular resistance (CVR), from 2.91 +/- 0.29 to 4.31 +/- 0.52 resistance units (RU) in the thalamus, from  59 +/- 5.0 to 47 +/- 5.0 ml/100 g per min (CVR: from 2.46 +/- 0.28 to 2.92 +/- 0.35 RU) in the hypothalamus, and from 21 +/- 1.0 to 17 +/- 0.8 ml/100 g per min (CVR: from 6.367 +/- 0.35 to 7.672 +/- 0.40 RU) in the white matter during ipsilateral stimulation. Contralateral stimulation of the sciatic nerves caused a more moderate but likewise significant drop in rCBF and an increase in CVR except in the white matter. Parallel to these changes, tissue aO2 decreased by 25 +/- 2% in the thalamic and by 19 +/- 2% in the hypothalamic area, relative to the prestimulation level.


Interorgan transport of amino acids in hemorrhagic shock. Arterial concentrations and net organ metabolism of amino acids (AA), O2, CO2, H+, and glucose (Glc) were measured in two dogs before and during hemorrhage and after blood replacement. Shock caused increased splanchnic and decreased peripheral blood flow and O2 consumption. Po2 decreased more in hepatic venous than in mixed venous blood. pH fell in hemorrhage and increased with retransfusion. Increased liver output and arterial concentration of Glc were observed during hemorrhage. Differences between animals correlated with nutritional status. Blood concentrations of most AA showed little change during hemorrhage but increased after retransfusion. In contrast, arginine concentrations declined sharply. Peripheral output and hepatic uptake of most AA occurred during the control period. During shock, peripheral output and hepatic uptake of total AA and most individual AA declined progressively; after retransfusion peripheral uptake and hepatic output of many AA occurred. By contrast, peripheral output and hepatic uptake increased for alanine, glutamine, serine, phenylalanine, and tyrosine. After retransfusion net transport of some compounds occurred from periphery to liver; others, from liver to periphery. During shock, hepatic protein catabolism increased. and this catabolism, accompanied by decreased hepatic uptake (increased hepatic output), seemed the main cause of increased blood AA concentrations. Protein catabolism in peripheral tissue was not a cause of increased blood concentrations. Pathological changes in pH, Po2, and blood flow, occurred early in hemorrhage. In contrast, changes in AA movements and concentrations were within normal limits until late in shock.


Effect of the histamine (H2) inhibitor metiamide on histamine-stimulated bile flow in dogs. The effects of the histamine H2-receptor inhibitor metiamide on histamine-stimulated canine bile flow and gastric hydrogen ion output were evaluated. Histamine was found to stimulate bile volume in doses comparable to those that stimulated gastric hydrogen ion output; both responses appeared to have the same maximal response dose, 150 mug/kg per h. Metiamide alone did not alter hepatic bile flow. Administration of metiamide, 2 mg/kg per h, along with various doses of histamine demonstrated that the H2-receptor antagonist decreased bile volume and gastric hydrogen ion output from values obtained with histamine administration alone. The D50 of histamine for bile flow was 16.3 mug/kg per h and the D50 for hydrogen ion output was 44.2 mug/kg per h, Kinetic analysis suggests that the decrease in histamine-stimulated hydrogen ion output produced by metiamide is the result of competitive inhibition; the decrease in histamine-stimulated bile volume by metiamide which is different from the hydrogen ion inhibition, suggests noncompetitive inhibition. These data indicate that the mechanism of histamine choleresis is different from the mechanism of histamine-stimulated gastric acid output and that histamine-stimulated bile flow may not be the result of direct hormone-receptor interaction.


Sympathetic nervous system and renin release from submaxillary glands in vitro. We previously reported that alpha- but not beta-adrenergic agonists stimulate renin release from mouse submaxillary glands in vivo. The present studies were undertaken to determine if these in vivo effects were due to a direct action on the submaxillary glands and to find out if cyclic AMP (cAMP) might be involved in submaxillary renin release. Pooled mouse submaxillary gland slices were incubated in Krebs-Ringer bicarbonate medium following a preincubation period, and renin release was measured by a radioimmunoassay for the direct measurement of submaxillary gland renin. Tissue cAMP levels were also measured. Addition of the alpha-adrenergic agonists, phenylephrine or norepinephrine, significantly increased renin release (P less than 0.01 vs. control) while decreasing tissue cAMP levels (P less than 0.01 vs. control). In contrast, addition of the beta-adrenergic agonist isoproterenol markedly increased cAMP levels (P less than 0.01 vs. control) and decreased renin release (P less than 0.05 vs. control). Pretreatment of the slices with the alpha-blocker phenoxy genzamine inhibited the effect of phenylephrine. These results indicate that alpha-adrenergic agonists cause renin release from submaxillary glands which is accompanied by a fall in tissue cAMP levels. This is in contrast to renin release from the kidney which is stimulated by beta-adrenergic agonists.


H+ current response to CO2 and carbonic anhydrase inhibition in turtle bladder. To evaluate the role of CO2 and carbonic anhydrase (CA) in H+ transport (JH) by turtle urinary bladder the effect of CO2 addition, with and without addition of CA inhibitiors, was examined on JH. Since in the presence of exogenous CO2 and HCO3- the pH stat-measured rate of mucosal (M) acidification underestimates JH by the rate of electroneutral HCO3- secretion, the reverse short-circuit current (RSCC) applied across ouabain-treated bladders was used to estimate JH. That the RSCC is a measure of JH was demonstrated by: 1) in the absence of added CO2 and HCO3- the rate of M acidification totally accounted for the RSCC, and 2) increases in RSCC with CO2 addition occurred without changes in Na+ and K+ fluxes or the coupled ration of HCO3- secretion for Cl-absorption. When serosal (S) percent CO2 was progressively progressively increased JH achieved a maximum rate of 64 +/- 3 muA (SE) with 4.5% CO2. At higher S percent CO2 JH did not change, suggesting that factors other than the rate of CO2 hydration were rate limiting. The maximum rate of JH was not decreased by low concentrations of CA inhibitors (acetazolamide, 5 X 10(-5) M), although the percent CO2 at which this maximum rate occurred increased to 8.5%. The increased percent CO2 requirement for the maximum rate of JH with low concentrations of CA inhibitors suggests that these agents alter JH by decreasing the rate of enzymatic CO2 hydration. At high concentrations (acetazolamide, 5 X 10(-4) M) these inhibitors decrease the maximum rate of JH in the presence of CO2, implying that these inhibitors at higher concentrations directly interfere with the H+ transport system.


Effect of potassium depletion on cerebrospinal fluid bicarbonate homeostasis. We have examined the effect of K depletion on CSF [HCO3-] homeostasis in awake rats. The relationship of CSF [HCO3-] to arterial [HCO3-] in metabolic acid-base disturbances is displaced is an upward direction and has a significantly increased slope in K-depleted vs. control rats (0.51 +/- 0.02 vs. 0.42 +/- 0.02). Results of partial K-repletion experiments, with peripheral acid-base balance held constant, suggest that the effect is K specific. The K-depleted animals also exhibit a wider (CSF-arterial) PCO2 difference than controls (11.1 vs. 8.4 mmHg). When CSF [HCO3-] is shown as a function of CSF PCO2 the data of K-depleted rats are no longer displaced when compared to controls but still have a significantly greater slope (1.21 +/- 0.23 vs. 0.89 +/- 0.08). This increased slope is interpreted to reflect enhanced HCO3- movement from blood to CSF at high arterial [HCO3-]. Analysis of our data and observations from the literature in conditions of mixed acid-base disturbances suggest that CSF [HCO3-] is determined by a) CSF PCO2 and b) the level of arterial [HCO3-] when the latter is greater than the normal CSF [HCO3-].


Rational treatment for an irrational disorder: what does the schizophrenic patient need? The author discusses the results of a review of controlled studies of treatment approaches to schizophrenia. Although the research evidence strongly supports the efficacy of pharmacotherapy, this finding should not be interpreted as meaning that all schizophrenic patients should receive antipsychotic drugs or that other forms of treatment are unnecessary. The author cautions against doctrinaire attitudes and advocates thoughtful adjustment of goals and methods to meet the needs of the various parties and situations involved in the treatment of the schizophrenic patient.


Infective larvae of Brugia: escape from mosquitoes into water and subsequent oral infectivity in jirds. Published work showed that third-stage larvae (L-3s) escape into water from dead or dying, Brugia pahangi-infected, Aedes aegypti. The present study revealed the same escape phenomenon among B. pahangi-infected Armigeres subalbatus, Anopheles quadrimaculatus, and Aedes togoi, and among Brugia malayi-infected Ae. aegypti and Ae. togoi. L-3s maintained in water or in Lum's solution for 3 hours retained infectivity when tested in orally or subcutaneously exposed jirds; furthermore, L-3s recovered from mosquitoes dead for 24 to 48 hours were also infective by either portal of entry in jirds. Since L-3s may escape and remain infective in the field, it is conceivable that natural filarial infections might thus be acquired orally by definitive hosts.


Experimental studies on the transmission of hepatitis B by mosquitoes. Culex tarsalis and Aedes aegypti mosquitoes were fed on chimpanzees carrying hepatitis B surface antigen (HBS Ag) of known infectivity and pools were tested by radioimmunoassay daily for the presence of HBS Ag. HBS Ag continued to be detected at low levels in mosquito tissue after digestion of the blood meal. Inoculation of susceptible chimpanzees with macerated pools of A. aegypti mosquitoes at two intervals after digestion of the blood meal did not produce hepatitis or serologic evidence of hepatitis B virus infection. Mechanical transmission studies by interrupting feeding of A aegypti from HBS Ag-carrier chimpanzees and transferring them to susceptible chimpanzees did not produce hepatitis. These findings do not support the hypothesis that mosquitoes are involved in either biological or mechanical transmission of hepatitis B.


Plasma activities of lysosomal enzymes after hepatic dearterialization in man. Six patients with liver metastases from carcinoid or colon carcinoma underwent hepatic derterialization. This operation, known to cause both tumor necrosis and liver cell damage, caused considerable increases of several lysosomal acid hydrolases in the circulation. Thus, beta-glucosidase showed a small temporary increase during the operation, followed by a slower but higher reaction reaching a maximum 12 to 36 hours postoperatively. Similar reactions were noted for beta-glucuronidase, acid phosphatase, beta-galactosidase, arylsuphatase A, and N-acetyl-beta-glucosaminidase while no reactions were found for cathepsin D. Very high enzyme levels occurred in a patient dying from bleeding complications in the postoperative period.


[Combined acupuncture analgesia and controlled respiration. A new modified method of anesthesia in open heart surgery (author's transl)]. From October 1973 to May 1975 cardiae surgery with extracorporal circulation using Acupuncture as Analgesia was performed in 131 patients in the surgical clinic of the University of Giessen. Acupuncture is performed by electric-stimulation of 8 points at the lower legs, the neck and ears with 90 V and 5--6 Htz. All patients are intubated; controlled respiration is maintained with 50% O2 and 50% N2o. During the whole operation, 20 min after intubation the patients are responsible. The main advantage of the new method we see in a good postoperative cooperation of the patients, which are generally extubated in the operation-room.


On the morphology of the human Sertoli cells under normal conditions and in patients with impaired fertility. Sertoli cells in testicular biopsies from 7 patients with Sertoli-cell only syndrome, 8 patients who had been treated with cyclophosphamide for testicular neoplasia, 8 patients with oligozoospermia, 7 patients with cryptorchidism, and 8 patients with seminoma were examined under the electron microscope and compared with Sertoli cells of normal tissues. The investigations reveal that each of these conditions of impaired fertility is characterized of a special type of Sertoli cell. The cell pattern is either restricted to one or two of the normally occurring cell types, or modified cells predominate that might be typical of the underlying disturbances of spermatogenesis. It is suggested that the cell type which prevails in the Sertoli-cell only syndrome is capable of maintaining a basic production of a substance that inhibits FSH secretion.


Interaction of anesthesia, beta-receptor blockade, and blood loss in dogs with induced myocardial infarction. The cardiovascular effects of halothane-nitrous oxide anesthesia, and beta-receptor blockade with either propranolol or practolol, were studied in 15 dogs in which severe myocardial infarction had been induced ten days earlier. The hemodynamic responses to blood loss amounting to 25 per cent of estimated blood volume, and its subsequent replacement, were studied before and after induction of beta-receptor blockade. In terms of cardiac output and aortic blood flow acceleration, cardiac performance in the absence of beta-blockade was markedly impaired during steady-state anesthesia, compared with corresponding values in normal dogs. Practolol (2.0 mg/kg) administered during anesthesia induced no significant circulatory change other than a 14 per cent decrease in heart rate and a 25 per cent increase in strode volum. Propranolol (0.3 mg/kg) caused a comparable reduction of heart rate, but significantly reduced cardiac output (-27 per cent), aortic blood flow acceleration (-26 per cent), and peak LV power (-19 per cent), and increased systemic vascular resistance (+49 per cent). The two drugs caused comparable shifts of the isoproterenol dose-response curve during anesthesia. Graduated blood loss during anesthesia, to a total of 25 per cent of blood volume, caused consistent circulatory changes (decreased mean arterial pressure cardiac output, peak LV power, LV minute work) that were essentially similar before and after beta-receptor blockade with either propranolol or practolol. The positive inotropic effect of calcium gluconate during halothane anesthesia was significantly reduced following either propranolol or practolol, but the hemodynamic responses to changes of systemic vascular resistance induced with acetylcholine or phenylephrine were not modified by beta-receptor blockade.


Characterization and tissue distribution of N-acetyl hexosaminidase C: suggestive evidence for a separate hexosaminidase locus. 1. An electrophoretic system in which N-acetyl hexosaminidase C (HEX(C)) MIGRATES LESS ANODALLY THAN N-acetyl hexosaminidase A (HEX(A)) is described. 2. HEX(C) is shown to differ from HEX(A) and HEX(B) in substrate specificity, molecular size and affinity for Concanavalin-A. 3. HEX(C) is present in a wide range of adult and foetal tissues and in tissues from patients with Tay-Sachs and Sandhoff's diseases. It is particularly prominent in brain, testis, thymus and lymphoblastoid cell extracts and in several foetal tissues. 4. It is suggested that HEX(C) is coded at a separate gene locus from HEX(A) and HEX(B).


Monitoring the administration of methotrexate in antimetabolite therapy. An enzymatic method for the measurement of methotrexate (MTX) in serum is presented in which the inhibition of the enzyme dihydrofolate reductase by MTX is measured. Reduction of the substrate dihydrofolate by the enzyme and cofactor NADPH is lessened in direct proportion to the amount of MTX present. Measurements can be made in the "therapeutic range" of MTX which corresponds to the 10(-7) to 10(-8) M concentration of MTX in serum.


Stool and urinary sugars in normal neonates. The pH of the stool and the amount of reducing substances present were observed in 51 normal neonates aged 5 to 8 days. A stool pH of 5 or less was found in 6, 4 of whom were exclusively breast fed. Reducing substances, 0-5% or more, were found in the stools of 16. Stool chromatography in 13 showed lactose, glucose, galactose, or a variable combination of these sugars--that is, a pattern consistent with lactose malabsorption. The stools of 3 infants contained oligosaccharides or maltose only. Chromatography of urine from 60 normal neonates showed detectable sugars in 11 but only 3 had levels above 50 mg/100 ml.


HCG stimulation test in children with abnormal sexual development. Plasma testosterone was estimated by radioimmunoassay in 60 children with disorders of sexual development before and after stimulation with human chorionic gonadotrophin (HCG). In 21 children the testosterone levels after 3 and 5 daily injections of 1000 units HCG were compared and good correlation was found between the paired results (r =0-93), suggesting that the 5-day HCG test has no advantage over the 3-day test. In 7 boys with apparently normal genital development the increments in plasma testosterone ranged from 2-0 to 8-5 nmol/1 after 3 injections of HCG. 10 boys with anorchia showed little response to HCG stimulation, but in patients with other disorders, such as micropenis (10), cryptorchidism (8), hermaphroditism (3), male pseudohermaphroditism (13), hypospadias (3), and sex chromosome anomalies (6), there was considerable variation in the plasma testosterone level after HCG. In 2 boys with suspected anorchia the results suggested that testes were present and this was confirmed at operation.


Human skin proteases. Fractionation of psoriasis scale proteases and separation of a plasminogen activator and a histone hydrolysing protease. Psoriatic scale proteases were found to be extracted effectively in salt solution (1 mol/l) containing Triton X-100 (5 g/l). The extraction in dilute buffer or sucrose yielded low activities. The acid (0.25 N H2SO4) and KSCN (2 mol/l) solutions effectively extracted plasminogen activator. Fibrinolysin was most active in salt (1 mol/l KCl) and in KSCN (2 mol/l) extracts. Psoriatic scale proteases were fractionated by Sephadex G-100 gel filtration and further by DEAE cellulose chromatography. Five different enzyme preparations were obtained. The first preparation, resembling cathepsin D, effectively hydrolysed hemoglobin at pH 3.5 and casein at pH 5.8 and was insensitive to protease modifiers. The second preparation effectively hydrolysed trypsin substrates (AGLME, TAME, BAEE and BANA) and also histone and casein at pH 7.2 and was inhibited by protease inhibitors, TLCK and E-600. The third preparation hydrolysed histone and casein at pH 10.2 and was effectively inhibited by E-600 and partially by protease inhibitors and TPCK. The fourth preparation, resembling cathepsin B1, hydrolysed BANA and BAEE at pH 5.8 and was activated by SH-reagents and EDTA. The fifth enzyme preparation hydrolysed ATEE and was inhibited by E-600 and TPCK. Plasminogen activator was found mainly in the second enzyme preparation and fibrinolysin activity in the third and fifth enzyme preparations. The second, third and fifth enzyme preparations were different from the enzymes found in healthy human skin. The proteases of psoriatic scale resemble those of tissue and cell cultures undergoing rapid cell division. The possible role of proteases in the increased cell division in psoriasis plaque is discussed.


Sympathomimetic-induced effects in the soleus muscle of the guinea-pig. Sympathomimetic amines reduced the peak tension, time to peak and time to half-relaxation of indirectly elicited twitches of the guinea-pig soleus muscle in vivo. Clonic contractions of the soleus muscle were depressed by the amines. On a molar basis salbutamol and orciprenaline were 5.12 and 80.0 times less potent than (+/-)-isoprenaline in producing these effects. Results obtained with the beta-receptor antagonists propranolol, practolol and H35/25 suggest that the depression in skeletal muscle contractility is due to beta2-adrenoreceptor stimulation. The effects of the amines on twitches and clonic contractions of the guinea-pig soleus muscle are qualitatively similar to those reported previously in the cat soleus muscle preparation, which has been used to assess the possible tremorogenic actions of sympathomimetic bronchodilators. On a quantitative basis the molar dose-ratios of the sympathomimetics used, and the effects of beta-receptor antagonists on the responses, are similar in the two species.


The effects of the beta-adrenoceptor blocking agent LL 21-945 on renal hypertensive (Grollman) rats. Prophylactic administration of LL 21-945 [4-(3-tert. butyl-amino-2-pivaloyloxypropoxy)-9-fluorenone] during 15 weeks to Grollman rats depressed the development of hypertension, tachycardia and myocardial pathogical changes. The tachycardia was eliminated and the degree of the myocardial pathological changes and coronary sclerosis was less severe than in the untreated Grollman rats. The survival rate was slightly improved with LL 21-945. With respect to the biochemical parameters studied, only a tendency of LL 21-945 to moderate the rises in blood cholesterol, total glycerides and urea was observed towards the end of the experiment.


Mesenteric vasodilator effect of 5-hydroxytryptamine: possible enteric neuron mediation. The superior mesenteric blood flow response to intra-arterial injections (0.5-25 mug) and infusions (5-30 mug/min) of 5-hydroxytryptamine (5-HT, serotonin) was investigated in anesthetized cats in which nerve activity to the intestine was altered by surgical and pharmacological procedures. With the superior mesenteric periarterial nerves intact, low doses of 5-HT (less than 5 mug) produce vasodilatation, whereas higher doses produce vasoconstriction. When the periarterial nerves are cut either at the start of or during the experiments, vasodilatation is elicited over the entire dose range, and doses of 5-HT which initially produce vasoconstriction elicit vasodilatation after nerve sectioning and also after alpha-adrenergic receptor blockade. These vascular responses are not secondary to changes in arterial pressure or intestinal motility. The vasodilator response to 5-HT is unaffected by alpha- or beta-adrenergic or cholinergic receptor blockade, by ganglionic blockade, or by histamine receptor blockade, but is blocked by tetrodotoxin and also the 5-HT antagonist, dihydroergotamine.


Electrophysiologically determined quinidine-like actions of a beta-adrenergic blocking agent, 1-(7-indenyloxy)-3-isopropylaminopropane-2-ol hydrochloride (YB-2). Since 1-(7-indenyloxy)-3-isopropylaminopropane-2-ol hydrochloride (YB-2) has been reported to be a potent beta-adrenergic blocking agent with antiarrhythmic effects, the electrophysiological effects of this compound on guinea-pig and canine heart muscle were examined. YB-2 was found to have typical quinidine-like actions such as decrease in rate of rise of the action potential, prolongation of refractory period and decrease in conduction velocity.


The effect of trimepranol on the binding of [3H] norepinephrine in myocardial subcellular fractions of the dog. The effect of Trimepranol on the binding of 3H norepinephrine to myocardial particles from dogs (to fractions 1,000 g and 78,000 g resp.) has been studied and compared with the effect of propranolol, isoprenaline, phentolamine and ephedrine. Displacement of 3H norepinephrine by propranolol and isoprenaline was found in both fractions. Trimepranol, in low concentrations, inhibited the binding of 3H norepinephrine; in higher concentrations the binding of 3H norepinephrine was stimulated. Phentolamine and ephedrine were without effect in both fractions. The problem of specificity of adrenergic binding sites is discussed.


Regional variation in the distribution of alpha-adrenoreceptors in the vas deferens of the rat. Experiments were performed with preparations obtained by medial transection of the rat vas deferens. Eight sympathomimetic drugs: noradrenaline, alpha-methylnoradrenaline, phenylephrine, dopamine, tyramine, amphetamine, cocaine and isoprenaline, were applied to preparations which were field-stimulated at 10 sec intervals. On the testicular half of the tissue all except isoprenaline enhanced twitches and/or caused contractions. These effects were blocked by alpha-adrenoreceptor antagonists. On the urethral preparation all except phenylephrine, which often enhanced twitches, inhibited responses to field stimulation. These effects were not blocked by alpha-adrenoreceptor antagonists or by propranolol, though the latter blocked inhibitory effects of isoprenaline on both segments. Inhibitory responses to tyramine persisted in urethral segments treated with cocaine and in urethral segments taken from reserpine-treated rats, and thus tyramine may have some direct action. No regional variation was observed in response to acetylcholine or carbachol. High potassium concentrations were equally effective in contracting both segments, but the time courses of contractions differed. These experiments confirm a density gradient of excitatory alpha-adrenoreceptors along the length of the rat vas deferens.


Studies on the action and interaction of dopamine and prostaglandin A1 in the renal vasculature. Dopamine (3 mug/kg/min) and prostaglandin A1 (0.2 mug/kg/min) were found to dilate the renal vasculature and increase total kidney blood flow in be anesthetized dog. These effects of dopamine, but not of prostaglandin A1, were completely antagonized by bulbocapnine, a selective dopamine receptor inhibitor, at a dose (3 mg/kg) which did not itself significantly alter cardiovascular hemodynamics. Conversely, indomethacin, an inhibitor of prostaglandin synthetase in the dog at 2 mg/kg, did not reduce the dopamin renal vascular response. These results suggest that dopamine and PGA1 decrease renal vascular resistance in the dog via distinct pharmacological mechanisms.


Morphological features of red blood cells in subjects with sickle cell trait: changes during exercise. Occasional complications and even death in subjects with sickle cell trait have been attributed to severe physical exertion. However, the extent to which sickling actually occurs during exercise has not been reported. This study examined the red blood cell morphological features immediately following near maximal upright graded bicycle exercise in five asymptomatic black subjects with hemoglobin AS. Exercise produced minimal sickling in vivo, which was not proportional to the intensity of exercise. The amount of sickling in vivo was small in comparison to that observed in the presence of severe hypoxia in vitro, never exceeding 0.75%. in seven normal subjects with hemoglobin AA, exercise did not cause changes in red blood cell morphological features. We conclude that exercise may initiate sickling in subjects with sickle cell trait.


Lactic acidosis in diabetic patients.

Lactic acidosis in diabetic patients. Plasma lactate and beta-hydroxybutyrate concentrations were measured during episodes of ketoacidosis and lactic acidosis in diabetics. In 39 patients with ketoacidosis, with a mean plasma beta-hydroxybutyrate concentration of 12.4 millimols/liter, the plasma lactate concentration was less than 3.6 millimols/liter in 28 and moderately elevated in 11. In seven of the latter, coexisting lactic acidosis had been suspected clinically. In these 39 patients, there was no correlation between plasma lactate and beta-hydroxybutyrate concentrations. In six of ten episodes of presumed and later proved lactic acidosis, despite negative or weakly positive serum reactions with sodium nitroprusside, the plasma beta-hydroxybutyrate concentration was elevated. Although positive, the serum reaction with nitroprusside was also misleadingly weak in five of 39 patients with ketoacidosis, including three with plasma lactate concentrations less than 3 millimols/liter.


Effect of the pH of culture medium on the alkalophilicity of a species of Bacillus. The amino acid incorporation and alpha-amino-isobutyric acid (AIB) uptake of an alkalophilic Bacillus grown at pH 8.2 (the pH 8-bacteria) were much less pH dependent (less alkalophilic) than those of the organisms grown at pH 10.0 (the pH 10-bacteria), respectively. The rate of AIB uptake of the pH 10-bacteria was almost the same as that of the pH 8-bacteria, while the rate of amino acid incorporation of the pH 10-bacteria was higher than that of the pH 8-bacteria in alkaline environments. The colloidal titration with clupein showed that the amount of negative charge on the pH 10-bacteria was greater than that of the pH 8-bacteria in alkaline environments. Considerable difference in protein composition was observed between the membranes of the pH 8- and 10-bacteria while no difference was observed in phospholipid composition.


[Mg dependence and other properties of fructose-1, 6-diphosphatase and glucose-6-phosphatase in various organs of cattle]. The properties of fructose-1.6-disphosphatase in supernatant of homogenate of liver, kidneys, and adductor muscles of cattle were tested. EDTA was found to activate the enzyme up to concentrations of 10 mMol. The pH optimum was 7.5 in the presence of EDTA. Even lower concentrations of magnesium ions caused activation of the enzyme, but an activating effect was obtained as well from relatively high Mg concentrations. Fructose-1.6-diphosphatase could by activated also by 0.2 mMol Mn or Co ions or 1 mMol Zn ions. Inhibitive action was obtained from Cu, Cd, Pb, and Hg ions. Microsomal fractions of cattle liver and kidney caused high activity of glucose-6-phosphatase, but only low action was obtained be using microsomal fractions of mesenteric mucous membrane or brain. Mg ions, basically, failed to trigger activation, and higher concentrations even caused inhibition. The relatively high activity of both fructose-1.6-diphosphatase and glucose-6-phosphatase in kidney of cattle appeared to suggest an involvement of those enzymes in gluconeogenesis.


Specific gamma-glutamyl transpeptidase inhibitor from human and animal intestines. An inhibitor of gamma-glutamyl transpeptidase in human and animal intestines was assayed by means of a new method. In mice fed with LSM fodder, accumulation of the inhibitor in the mucous membrane of the small intestine was observed. Purified gamma-glutamyl transpeptidase inhibitor from mouse and human intestines was identified as L-serine. Neither this amino acid nor purifed inhibitor acted on other intestinal peptidases. Presumably, one of the ingredients of LSM feed influences accumulation of the inhibitor, and consequently diminishes absorption of gamma-glutamyl substrates in the intestine.


Specificity and inhibition of gamma-glutamyl transpeptidase in guinea pig intestines. Gamma-Glutamyl transpeptidase activity in guinea pig intestine was tested toward 8 monoglutamyl and 4 diglutamyl substrates. Distinct differences in specificity and activity related to ontogenetic development were noted. A mixture of L-serine and borate inhibited specifically gamma-glutamyl transpeptidase activity of homogenates and accumulation of naphthylamine and 14C-substances in slices of intestine incubated with gamma-L-14C-glutamyl-alpha-naphthylamide. The same mixture, administered per os together with 14C-gamma-glutamyl substrate, specifically inhibited urinary excretion of 14C-substance, but had no effect on the rate of excretion of glycine, L-glutamic acid or L-pyroglutamic acid.


Simultaneous determination of glutethimide, methyprylon, and methaqualone in serum by gas liquid chromatography. A gas chromatographic method is described that permits the simultaneous determination of glutethimide, methyprylon, and methaqualone in serum samples. The threshold of sensitivity for each of the three hypnotics is 0.2 mg/1.


Determination of lorazepam in plasma by electron capture GLC. A method is described for the determination of lorazepam plasma levels involving extraction from the sample and analysis of the intact lorazepam by electron capture gas-liquid chromatography. Using mass spectrometry it is demonstrated, that lorazepam shows a thermal rearrangement under gas chromatographic conditions. The limit of detection is 0.01 mg/l and the assay shows a linearity from 0.01-0.80 mg lorazepam per liter of plasma.


[Structure of bromine containing metabolites of carbromal (author's transl)]. Isolation of three bromine containing metabolites from human urine is described and their chemical structure is proved by comparison of their physical properties with those of synthetically prepared substances. 2-Bromo-2-ethyl-butyramide, a pharmacologically active product, is showed to be the most important bromine containing metabolite by volume. In 3-position hydroxylated metabolites of carbromal, 3-hydroxy-carbromal and 2-Bromo-2-ethyl-3-hydroxy-butyramide have same physical properties as the synthesized DL-threo--diastereomeres.


Some properties of togavirus hemagglutinin studied with the aid of kaolin-adsorbed virus. Acidification (at pH 5.75) of Semliki Forest virus and West Nile virus suspensions completely eliminated their hemagglutinating activity within several minutes, but did not affect their infectivity or change their ability to absorb homologous hemagglutination-inhibition antibodies. In order to assay antibody absorption it was necessary to remove all of the immune complex from the reaction mixture, because the immune complex inhibited additional hemagglutinin. Removal of the immune complex can be accomplished by the use of kaolin-absorbed virus. This procedure is simple and dependable and has been carried out with viruses from several groups--Toga-, Myxo-, Paramyxo- and Picornaviridae.


Pyridine nucleotide metabolism in imaginal discs of Drosophila melanogaster. The pyridine nucleotide metabolism of imaginal discs of Drosophila melanogaster has been studied in vitro by incubating discs with labeled nicotinic acid in the presence and absence of ecdysterone. The major labeled compounds found within the discs are NAD, NADP, and nicotinic acid. There is preferential uptake of nicotinamide over nicotinic acid, although the Priess-Handler pathway is used exclusively. The presence of ecdysterone produces a small increase in the NADP/NAD ratio, and an increase in NAD synthesis, probably to compensate for increased NAD turnover.


Glucose 6-phosphate dehydrogenase in rainbow trout. Electrophoretic analysis of glucose 6-phosphate dehydrogenase from liver and blood of rainbow trout revealed a complex series of bands, which could differ between fish. The partial interconvertible nature of these bands was demonstrated with enzyme that had been incompletely inactivated at pH 8.4. In a single population of 40 fish, a homozygote and a heterozygote for an electrophoretic variant allele were found. We suggest that G6PD in rainbow trout liver and blood is determinted  by two alleles at a single locus, with posttranslational modification responsible for the complex electrophoretic patterns seen. The basis for this variation appears to be NADH binding to the protein molecule. Another variant and other properties of the enzyme are described.


Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose. Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.


The nature of the residual alpha-mannosidase in plasma in bovine mannosidosis. Acidic alpha-mannosidase (EC 3.2.1.24), optimum pH 4.25, is absent from the plasma of Angus calves with mannosidosis, and the residual alpha-mannosidase activity has an optimum pH of 5.5, intermediate between that of the acidic and neutral alpha-mannosidases. This 'intermediate' alpha-mannosidase differs from the acidic form in its kinetic properties, its lack of marked inhibition by EDTA and its thermolability at 55 degrees C and physiological pH. Isoelectric focusing and ion-exchange chromatography show that it exists in at least two forms. The presence of a secondary peak at pH 5.5 in the pH/activity profile of normal plasma and the effect of heating at 55 degrees C indicate that such a form is present in normal plasma. The residual activity in the plasma of a calf with mannosidosis is therefore probably not the product of the defective gene. A differential assay, based on their different stabilities at 55 degrees C, has been developed for measuring the acidic and intermediate alpha-mannosidases in plasma. There was no correlation between the concentrations of the two enzymes in the plasma of Angus cows heterozygous for mannosidosis or in the plasma of normal animals. This precludes the use of the intermediate form as a reference enzyme for the acidic activity in a test for heterozygosity for mannosidosis based on the gene-dosage phenomenon. The concentrations of the intermediate activity were comparable in normal animals and animals homozygous or heterozygous for mannosidosis.


Iron-dependent binding of 8-anilinonaphthalene-1-sulphonate by both lactoferrin and transferrin. Fluorescence of 8-anilinonaphthalene-1-sulphonate is enhanced by both lactoferrin and transferrin. The enhancement is relatively low between pH 4 and 8, and high at below pH 4. At physiological pH the fluorescence enhancement is higher with the iron-deprived than with the iron-saturated proteins. Binding of iron by lactoferrin is associated with lowering affinity for the dye.


The kinetics of formation of horseradish peroxidase compound I by reaction with peroxobenzoic acids. pH and peroxo acid substituent effects. 1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.


Preparation of immobilized baker's-yeast glucose 6-phosphate dehydrogenase attached to modified sepharose and sephadex and a comparison of the properties of these preparations with those of the soluble enzyme. 1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.


Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases. 1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.


The beta-glucosidase in the gut contents of the snail Achatina achatina. 1. The enzyme beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from the gut contents of active Achatina achatina exists in two molecular forms, beta-glucosidase C (mol.wt. about 82000) and D (mol.wt. about 41000). 2. Only the lower-molecular-weight species was found in the gut contents of aestivating snails or in extracts from their digestive glands and washed gut walls. 3. On re-activation of some aestivating snails, betion of ATP and Mg2+ to the isolated gut contents or to extracts from washed gut walls led to the formation of higher-molecular-weight forms of the enzyme, beta-glucosidase A (mol.wt. about 329000) and beta-glucosidase B (mol.wt. about 165000). 5. All these forms of the enzyme have similar pH optimum (pH 5.0-5.6). 6. The Michaelis constants (Km) and heat stability of the enzyme increased with increasing molecular complexity.


Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker's-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose. 1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.


Kinetic studies on pantothenase from Pseudomonas fluorescens. Effects of pH on substrate and inhibitor binding. The velocity of the pantothenase-catalysed hydrolysis of pantothenate was studied over pH5.5-9, and in the presence of oxalate or oxaloacetate as an inhibitor. The pH-dependence of the reaction can be described by a kinetic equation containing two ionizations of the enzyme, with one ionizable group located at the substrate-binding site, and the other at the inhibitor-binding site. The Km value of pantothenase to pantothenate depends on the buffer used, and phosphate tends to give somewhat lower values than other buffers. Km also depends on pH, the best activities being observed at basic pH values. The pH-independent Km is 7.6mM in phosphate buffer at 20 degrees C; the corresponding Kapp.m value at pH7 is 15 mM. The pK value of the ionizable group at the substrate-binding site was measured by two methods: from the pH-rate profile and from the pH-Km rofile. pK is 7.0 in phosphate buffer at 20 degrees C, ranging in various buffers between 6.9 and 7.3. The van't Hoff enthalpies of substrate binding and H+ ion binding were--14kJ/mol respectively. The inhibition by oxalate or oxaloacetate is of non-competitive type and depends on pH, the inhibitors being effective at acidic pH values. The pK value of the ionizable group at the inhibitor-binding site was derived from the measurements of the K1 values over the pH range 6-7.5. The pK value was 6.4 in oxaloacetate inhibition, the pH-independent K1 being 0.36mM, and the corresponding Kapp.m about 1.8mM at pH7. Phenylmethanesulphonyl fluoride was capable of inactivating pantothenase.


The V-region sequence of the H chain from a third rabbit anti-pneumococcal antibody. The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.


Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups. Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.


The interaction of reduced nicotinamide--adenine dinucleotide phosphate with reduced nicotinamide--adenine dinucleotide--ubiquinone reductase from bovine heart mitochondria. Reduction of the chromophores of mitochondrial NADH-ubiquinone reductase by NADPH reaches only 50% of the extent of reduction by NADH, monitored at 450 nm. This effect is due to autoxidation of an enzyme component at a higher rate than its reduction by NADPH.


Haemolysis induced by tyrosine crystals: Modifiers and inhibitors. Tyrosine as a solid, but not in solution, caused human erythrocyte haemolysis. Haemolysis was increased with higher tyrosine concentrations and extended incubation times; it was greater at 37degrees than 4degreesC, and decreased by higher erythrocyte concentrations. Titration of phenolic groups on the surface of di-iodotyrosine crystals altered the extent of di-iodotyrosine-induced haemolysis. Haemolysis induced by tyrosine was inhibited by polyethylene glycol (mol.wt. 6000 or 20000) in a competitive fashion; polyoxyethylene/polyoxypropylene non-ionic detergents, polyvinylpyrrolidone (mol.wt. 40000 or 360000), 0.25--1.0M-NaC1, 0.25--1.0 M-KC1 and 0.25 M-NaSCN also inhibited haemolysis. H+-ion donation from the phenolic groups of tyrosine is suggested as part of the mechanism of haemolysis. Non-ionic detergents may inhibit tyrosine-crystal-induced haemolysis by binding the phenolic groups at the surface of the crystal.


Studies on the amino acid-incorporating activity of native rat liver rough membrane and that reconstituted in vitro. The amino acid-incorporating activities of free polyribosomes, rough membranes and rough membranes reconstituted in vitro, derived from rat liver, were compared. The amino acid-incorporating activity of the two membrane fractions were very similar in their response towards changes in pH, Mg2+ concentration and temperature, but differed from the response of the amino acid-incorporating activity of free polyribosomes. Free polyribosomes irreversibly lost part of their amino acid-incorporating capacity after they had become bound to rough membrane, from which the original ribosomes had been removed. Ribonuclease activity present in the membrane fraction may be responsible for this loss.


Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid. The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.


Characterization of the binding of human growth hormone to microsomal membranes from rat liver. The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.


Some aspects in the pharmacology of diclonium bromide (2-(3,2-dichloroanilino)quinolizinium bromide). Part I: Antispasmodic action. Diclonium bromide (EU-2972; 2-(3,4-dichloroanilino)quinolizinium bromide) was demonstrated to possess an effective, prolonged inhibitory action on contractions in response to stimuli or propulsive movements in the lower bowel of the dog. The compound's antagonism to contractile activity was greater in the distal colon than in the duodenum or upper bowel. Diclonium bromide was a nonselective antispasmodic but, unlike papaverine, caused profound antagonism against smooth muscle contractile responses to intrinsic motor neural excitation. The drug had weak specific anticholinergic action, but its lack of antagonistic effect on other cholinergic responses in this study indicates that the anticholinergic action contributes very little, if at all, to its overall antispasmodic effect. The compound has potential application in the treatment of spastic-colon disease.


[Antibacterial activity of sisomicin in comparison with gentamicin]. The antibacterial activity of sisomicin -- a new aminoglycoside antibiotic -- as compared with gentamicin was tested on 521 bacterial strains of different species in a serial-dilution test. Staphylococci, streptococci, E. coli, Klebsialla-Enterobacter, indole-psitive Proteus strains, pseufomonads, Salmonads, Salmonellae, and Serratia marcescens were inhibited to the extent of 100% at a maximun of 4.0 mug/ml. Sisomicin showed a higher antibacterial activity against part of the bacterial species. Gentamicin-resistant pseudomonads and Klebsiella (clinical isolates) were still inhibited to the extent of 42 and 67%, respectively, by sisomicin. In addition to the determination of the MIC values for the use of different liquid media, investigations on the determin ation of the minimal bactericidal concentration (MBC), the effect of serum, pH, and inoculum on the bacterial activity, and investigations on the resistance development in vitro were also carried out.


Association between HLA and cutaneous necrotizing venulitis. A group of patients has been identified with cutaneous necrotizing venulitis (vasculitis). These patients, some with concomitant connective tissue disorders, have skin lesions that  separate them from the arteritis commonly described as rheumatoid vasculitis. HLA typing has been performed on 31 of these unrelated patients with cutaneous necrotizing venulitis, including 19 with associated chronic disorders. The antigen pair A11, BW35 was found in 5 of these 19 patients and in 11 of 346 controls. This difference in frequency is statistically significant. Because HLA genes appear to be linked to immune response genes, these data suggest that such genes may exist in patients with this form of cutaneous necrotizing venulitis with associated connective tissue disease.


Studies on Vaccinium myrtillus anthocyanosides. I. Vasoprotective and antiinflammatory activity. A Vaccinium myrtillus anthocyanosides preparation (equivalent to 25% of anthocyanidins) demonstrated significant vasoprotective and antioedema properties in exerimental animals. In rabbits, the skin capillary permeability increase, due to chloroform, was reduced both after i.p. (25--100 mg/kg) and oral administration (200--400 mg/kg) of anthocyanosides. Their activity was more lasting in comparison to rutin or mepyramine and this did not seem to be due to a specific antagonism towards inflammatory process mediators such as histamine or bradykinin. Experiments carried out in rats demonstrated that Vacinium myrtillus anthocyanosides were effective both in skin capillary permeability test as well as on vascular resistance of rats fed a P factor deficient diet. In the former test effective doses were in the range of 25--100 mg/kg (by oral route). In both the animal species investigated, anthocyanosides were two-fold more active when compared to the flavonoid rutin. Vaccinium myrtillus anthocyanosides by oral route inhibited carrageein paw oedema in rats showing a dose-response relationship. An antioedema activity was detected also after i.v. or topical application.


Comparison of the action of BD 40 A and some other beta-adrenoceptor stimulants on the isolated trachea and atria of the guinea pig. 3-Formylamino-4-hydroxy-a-(N-1-methyl-2-p-methoxyphenethyl-aminomethyl)-benzylalcohol hemifumarate (BD 40A) was compared with isoproterenol, orciprenaline, trimetoquinol and salbutamol for its beta-adrenergic activity and selectivity in vitro. On trachea, the maximum relaxing responses to five agonists were similar, but the order of potency was BD 40A greater than trimetoquinol greater than isoproterenol greater than or equal to salbutamol greater than orciprenaline. On atria, the maximum chronotropic and inotropic responses to isoproterenol were greater than those to BD 40A, orciprenaline and trimetoquinol, and salbutamol caused the weakest cardiac stimulating effect. Namely, the latter four drugs appeared to be partial agonists on atria. Salbutamol showed the high selectivity for trachea, whereas orciprenaline and trimetoquinol were equipotent on trachea and atria. Isoproterenol was more potent on atria than on trachea. BD 40A had the highest bronchoselectivity among five agonists and seemed to act on the beta-adrenergic receptor directly.


The choice of neuroleptics in the treatment of schizophrenia: a critical review. The selective use of different neuroleptics in the treatment of schizophrenia by some psychiatrist according to the greater need for a sedative, antidelusional or activating effect is supported only by some uncontrolled clinical observations. Most extensive controlled studies done until now showed no significant differences among neuroloptics in their therapeutic efficacy. The hypothesis that these drugs have specific and selective actions therfore cannot be accepted. In the single patient it seems more rational to choose the right dosage schedule instead of the "right drug". Though the study of the metabolism of these drugs remains the most interesting approach to the problem of the individualization of the therapy., the results of these studies have until now been disappointing e.g. plasma levels of chlorpromazine correlated weakly with clinical improvement. In clinical practice an important element in the process of choosing is still the incidence of side effects and in fact at the moment "the drug of choice" can only be the drug best tolerated by the patient.


Interaction of psychotropic agents with central neurotransmitters as revealed by their effects on PGO waves in the cat. One of the phasic phenomena of REM (rapid eye movement) sleep, the ponto-geniculo-occipital (PGO) waves, are induced in cats by either depleting brain monoamines with the benzoquinolizine derivative Ro 4-1284 or inhibiting the synthesis of 5-hydroxy-tryptamine (5-HT) by p-chlorophenylalanine (PCPA). The effects of the most important psychotropic agents on PGO1284 and PGOPCPA are reported and explained by their interaction with one or more of the 4 neurotransmitters known so far to be involved in the regulation of the PGO wave generation in the pontine reticular formation. Tricyclic antidepressants depress PGO waves by inhibiting the neuronal uptake of norepinephrine (NE) and/or 5-HT. Some neuroleptics increase the density of GO waves by blocking 5-HT and/or NE receptors. Various indole hallucinogens depress PGO waves by stimulating 5-HT receptors. Benzoldiazepines appear to enhance a (gamma-aminobutyric acid)-ergic (GABA)-ergic inhibitory influence on NE neurons and increase the density of PGO waves in the presence of functionally intact NE neurons.


[Proceedings: Psychotropic drugs and quality of sleep: quantitative neurophysiological and subjective parameters (author's transl)]. The effect of drugs of the 3 main psychopharmaceutical classes (anxiolytics, neuroleptics, antidepressants) on objective and subjective sleep parameters was studied in healthy normal volunteers. Objective parameters included: computerclassified sleep stages, visually evaluated REM-activity as well as 22 variables of the quantitatively analyzed all-night sleep and REM-sleep EEG. Alterations of the subjective sleep quality were rated based on a sleep self-rating scale. It was found that anxiolytics, neuroleptics and antidepressants induce characteristic changes in the objective sleep parameters. Subjectively, the quality of sleep was best after anxiolytics, while the quality of awakening in the morning was dependent on the dose of the anxiolytic drug. Finally, the relationship between objective and subjective sleep parameters was explored.


[Interactions of dopamine receptor agonists and antagonists with regard to dopamine synthesis and metabolism]. Intraperitioneal injection of d-amphetamine sulfate, 0.3-3 mg/kg, led to a marked rise in dopa formation in the dopamine rich areas c. striatum and mesolibbic cortex of the rat brain inhibiton of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine HCL (NSD 1015). However, amphetamine given in a dose of 10 mg/kg decreased the tyrosine hydroxylation rate in the mesolimbic cortex as well as the norepinephrine containing neocortex. In combination with haloperidol the stimulating effect of the neuroleptic on dopa formation was markedly potentiated by amphetamine in rat forebrain. Also, amphetamine potentiated the haloperidol induced increase in dopamine release in vivo measured as 3-methoxytyramine formation. The functional antagonists haloperidol and d-amphetamine appear to have synergistic effects on dopaminergic neurons.


[Acute and chronic effects of carpipramine, clozapine, haloperidol and sulpiride on the metabolism of biogenic amines in the rat brain (author's transl)]. In acute and chronic experiments investigations were made concerning the effect of clozapine, haloperidol, sulpiride and carpipramine on MHPG, HVA and 5-HIAA in rat brain and on motor activity of the animals. The activity of the rats treated with clozapine and haloperidol was reduced on the first day. After 10 days of treatment this effected of clozapine was significantly diminished. The MHPG level increased slightly on the first day of treatment with all four drugs. This elevation was maintained after chronic treatment with carpipramine and sulpiride, whereas clozapine and haloperidol decreased the MHPG content. 5-HIAA values did not show significant changes in acute experiments, whereas in chronic ones there was an increase. Haloperidol and clozapine induced a strong increase of HVA which decreased after 11 days in those animals treated with haloperidol. In comparison to haloperidol, after clozapine application the percentage of HVA-increase was higher in the limbic system than in the nigrostriatum.


Stimulation of rat striatal tyrosine hydroxylase by phospholipids and adenosine-3',5'-monophosphate. Rat striatal tyrosine hydroxylase is stimulated in vitro by various phospholipids. This stimulation was produced by a 3- to 4-fold increase in affinity for pteridine cofactor. No change in the Km for tyrosine was observed, The sedimentation pattern of tyrosine hydroxylase on linear sucrose gradients showed no indication of enzyme dissociation in the presence of lysolecithin at maximal stimulatory concentration. Crude striatal tyrosine hydroxylase is also activated by a combination of ATP, Mg++, EGTA and cAMP. After removing these agents by Sephadex G-25 chromatography, the activated form of the enzyme can be further stimulated by lysolecithin. These results suggest a possible role for phospholipids in the regulation of striatal dopamine synthesis.


[Free description of drug effects and description by questionaire of a sleep inducer (flurazepam) by normal test subjects (author's transl)]. Giving flurazepam and placebo to students as subjects, the free description of drug-effects (FD) was compared to the description by a questionaire (QD). The results were: 1. The FD-method was more informative than the QD-method. 2. The FD-method induced less frequent placebo effects and more frequent verum-non-effects than did the QD-method. 3. The results (1) and (2) based mainly on the variables time-to fall-asleep, subjective quality of sleep and total time of sleep, in which variables flurazepam differed significantly from placebo.


[Personality-specfic action of a tranquilizer (author's transl)]. 23 emotionally lable and 22 emotionally stable subjects wefe selected from a total smaple of 147 students by three personality inventories (FPI, GT, MPI). In a 2(3)-factor design the following effects of a single dose of 1.5 mg bromazepam p.o. against placebo were found: whereas the fine-motor activity (tapping, line-tracing) was stabilized independently of personality traits, the performance in attention tests (d2, choice-reaction time) was decreased in the emotionally stable group. In the emotionally lable group, i.e. in those subjects for whom bromazepam could therapeutically be indicated, an advantageous effect on performance in these tests was possible. The following variables were not affected by personality or medication: after-image of spiral rotor, critical flicker fusion (CFF), taking-task


[Comparison of experimental-psychological and clinical findings on the effect of a test substance (author's transl)]. The results of a pharmacopsychological study on male students high or low in emotional stability are compared to those of a clinical study on neurotic out- and inpatients. These studies examine the effects of diazepam and various doses of a thienodiazepine (Bay g 5653), a drug under investigation. Although the studies are not completely comparable (placebo control missing in the clinical study, not enough information about comparable base line measures) the differences in effects of Bay g 5653 and diazepam on the actual emotional state, as measured by an adjective check list, show a certain amount of correspondence between normal subjects and patients but also considerable discrepancies.


[Changes in drug elimination under the influence of perazine therapy (author's transl)]. In 8 male patients who were treated with perazine for a schizophrenic psychosis (200-800 mg/die), the elimination rate of phenazone was investigated. Simultaneously determinations of plasma levels of perazine and desmethylperazine were carried out. The average half-life of phenazone was 27.0 h in perazine-treated patients and 11.2 h in controls. Correspondingly, the clearance of phenazone decreased from 47.0 ml/min to 18.9 ml/min under perazine, both differences being highly significant. The amount of 4-OH-phenazone, the principal hydroxylated metabolite excreted in the urine, was 66 mg/24 h in the perazine group, and significantly different from the results obtained in the control group: 185 mg/24 h. In contrast the urinary excretion of the unchanged phenazone increased from 29 to 40 mg/24 h under perazine. The results are interpreted to demonstrate inhibition of drug hydroxylation in the liver by perazine treatment.


Porcine malignant hyperthermia. III: Adrenergic blockade. The effects of the establishment of an adrenergic blockade on suxamethonium-induced porcine malignant hyperthermia (MH) were investigated in Pietrain pigs. Six animals were fed reserpine 10 mg daily for 7 days and then challenged with suxamethonium. Three survived but the remainder developed fatal MH. In a further study of 10 pigs, either phentolamine 40 mug/kg/min or propranolol 50 mug/kg/min were administered for 30 min before suxamethonium stimulation and continued for the duration of the experiment. The five year beta-blocked pigs all became hyperthermic and died whereas the phentolamine-treated group survived. However, both alpha adrenergic blockade and successful reserpinization failed to prevent the abnormal muscle response to the first dose of suxamethonium.


[Organization and results of cadaver kidney transplantation from 1969 to 1973 (author's transl)]. France transplant was founded in order to organize rationally the cadaver kidneys transport and transplantation with is omogenous compatibility tests. 2143 hemodialysis patients have been presently (1-9-72) treated in 82 dialysis center in France; 964 of them are in the France Transplant waiting list. According with recent laws, nervous function cessation is synonimous of death. That made possible, by good resuscitation techniques, to maintain a good level of circulation and oxigenation of organs. Family permit is required for this purpose. 17 medical transporttion staffs are at work in 12 France towns and cooperate with 12 typing laboratoires working with the same techniques and reagents. One permanent secretariat in Paris is always telex connected with all staffs and mantains a continuous up to date patients waiting list. 415 cadaver kidneys were transplanted, 255 in the same town and 160 trabsported from a town another. A significative rise in cold ischemia times happened recently because of the increasing number of transported kidneys.


Stimulation of the peripheral chemoreceptors with sodium bicarbonate. The i.v. administration of sodium bicarbonate was found to cause an increase in arterial pH, followed by an increase in PaCO2. This caused a large increase in lung ventilation in in PaO2. Oxygen administration in human subjects, and anatomical denervation of the chemoreceptors in dogs, caused a substantial delay in the ventilatory responses to sodium bicarbonate. It was concluded that the i.v. administration of sodium bicarbonate provides a method of testing the presence of peripheral chemoreflexes which has the advantage of being independent of alveolar ventilation.


Oral premedication with oxypertine.

Oral premedication with oxypertine. The preoperative effects of oral oxypertine have been compared with those of papaveretum and atropine in 185 patients in a double-blind between-patient study. Oxypertine 20 mg given orally as a nocturnal sedative and again on the morning of operation produced relief of anxiety comparable to that of papaveretum 10 mg and atropine 0.6 mg. It is concluded that oxypertine may be of value in medication before anaesthesia.


Formation of anhydrosugars in the chemical depolymerization of heparin. In the reactions used to break heparin down to mono- and oligosaccharides, androsugars are formed at two stages. The first of these is the well-known cleavage of heparin with nitrous acid to convert the N-sulfated D-glucosamines to anhydro-D-mannose residues; this reaction has been studied in detail. It is demonstrated here that only low pH (less than 2.5) reaction conditions favor the deamination of N-sulfated D-glucosamine residues; the reaction proceeds very slowly at pH 3.5 or above. On the other hand, N-unsubstituted amino sugars are deaminated at a maximum rate at pH 4 with markedly reduced rates at pH2 or pH6. At room temperature solutions of nitrous acid lose one-fourth to one-third of their capacity to deaminate amino sugars in 1 h at all pHs. A low pH nitrous acid reagent which will convert heparin quantitatively to its deamination products in 10 min at room temperature is described, and a comparison of the effectiveness of this reagent with other commonly used nitrous acid reagents is presented. It is also shown that conditions used for acid hydrolysis of heparin convert approximately one-fourth of the L-iduronosyluronic acid 2-sulfate residues to a 2,5-anhydrouronic acid. This product is an artifact of the reaction conditions, and its formation represents one of several pathways followed in the acid-catalyzed cleavage of the glycosidic bond of the sulfated L-idosyluronic acid residues.


Influence of substrates and coenzymes on the role of manganous ion in reactions catalyzed by pig heart triphosphopyridine nucleotide-dependent isocitrate dehydrogenase. The interaction of manganous ions with pig heart triphosphopyridine nucleotide (TPN) specific isocitrate dehydrogenase has been studied by kinetic experiments and by direct ultrafiltration measurements of manganous ion binding. At low metal ion concentrations, a lag is observed in the time-dependent production of reduced triphosphopyridine nucleotide (TPNH) that can be eliminated by adding 20 muM TPNH to the initial reaction mixture. A plot of 1/upsilon vs. 1/ (Mn2+) obtained at relatively high TPNH concentrations (20 muM) is linear and yields of Km value of 2 muM for metal ion, which is comparable to the direct binding constant measured in the presence of isocitrate. A similar plot at low TPNH concentrations (2 muM) reveals a biphasic relationship: at high metal concentrations the points are collinear with those obtained at high levels of TPNH, but at low metal concentrations that line is characterized by a Km of 19 muM for Mn2+. A difference in the deuterium oxide solvent isotope effect on Vmax observed with 20 muM TPNH as compared with 2 muM TPNH suggests that at high TPNH concentrations or high manganous ion concentrations the rate-limiting step is the dehydrogenation of isocitrate, while at low manganous ion concentrations and low TPNH concentrations, the slow step is the decarboxylation of enzyme-bound oxalosuccinate. Evidence to support this hypothesis is provided by the sensitivity to isocitrate concentration of the Km for total manganese measured in the presence of 20 muM TPNH that contrasts with the relative insensitivity to isocitrate of the Km measured at 2 muM TPNH and low manganous ion concentration. Direct measurements of oxalosuccinate decarboxylation reveal that the Vmax and the Km for manganous ion are influenced by the presence of oxidized or reduced TPN with the Km being lowest (5-7 muM) in the presence of TPNH. The dependence of the Km for manganous ion on the presence of substrate, TPN, and TPNH, is responsible for the variation with conditions in the rate-determining step. The enzyme binds only 1 mol of metal ion and 1 mol of isocitrate/mol of protein under all conditions. The pH dependence of the binding of free manganous ion, free isocitrate, and manganous-isocitrate complex indicates differences in the interaction of these species with isocitrate dehydrogenase. These results can be described in terms of two functions for manganous ion in the reactions catalyzed by isocitrate dehydrogenase, each of which requires a distinct binding site for metal ion: in the dehydrogenation step, Mn2+ facilitates the binding of the substrate isocitrate, and in the decarboxylation step it may stabilize the enolate of alpha-ketoglutarate which is generated.


Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells. The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.


Thyroid Ribonucleic Acid-Iodopeptides. Comparison of Tyrosyl-Complex II and Tyrosyl-tRNA. It has previously been shown that mammalian RNA-peptidyl complexes are found in close association with tRNA, but can be separated from the bulk of the tRNA by benzoylated diethylaminoethylcellulose chromatography (Kull, F.J., and Soodak, M. (1971), Biochim. Biophys. Acta 246, l; Gadski, R.A., and Kull, F.J. (1973), Biochemistry 12, 1907). These studies also showed that under aminoacylation conditions the complex fractions were able to act as acceptors for certain amino acids and that the formation of porcine thyroid tyrosyl-complex II was particularly high. Because of this high acceptor function, and because of the importance of tyrosine to thyroid metabolism, further studies were conducted comparing some of the properties of porcine thyroid tyrosyl-complex II with those of porcine thyroid tyrosyl-tRNA. Porcine thyroid tyrosyl-tRNA synthetase was purified in excess of 200-fold and characterized. It was found that maximal aminoacylation was achieved at pH 8.1 in the presence of 150 mM KCl. The Km for tyrosine was determined to be 3.0 X 10(-6) M. The purified thyroid tyrosyl-tRNA synthetase was used under aminoacylation conditions to prepare radioactively labeled porcine thyroid tyrosyl-tRNA and tyrosyl-complex II. Comparisons made using reversed-phase column chromatography (RPC-5) showed distinct differences between the two aminoacylated species and revealed, in addition, a number of isoaccepting forms of tyrosine tRNA. Tyrosyl-complex II was also found to differ from tyrosyl-tRNA in that it is more stable to deacylation at pH 7.0 and at pH 4.4 and to degradation by ribonuclease A. In addition, tyrosyl-complex II, unlike tyrosyl-tRNA, is degraded by trypsin. Ribosomal binding studies showed that tyrosyl-complex II did not respond to the codons for tyrosine, UpApU and UpApC, whereas tyrosyl-tRNA responded to both. It is suggested that thyroid tyrosine complex II is representative of a group of related complexes that constitute the complex II fraction and that, although the complexes resemble tRNA in many respects, they have distinctly different characteristics than conventional tRNA.


Molecular polymorphism and mechanisms of activation and deactivation of the hydrolytic function of the coupling factor of oxidative phosphorylation. The 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis has a latent adenosine triphosphatase (ATPase) function that can be activated by heating at 55 degrees C for 10 min at pH 8.5 in 50% glycerol. The specific activity increases from 0.1 to 20--30 mumol min-1 mg-1. Adenosine 5'-triphosphate (ATP) is not required for stabilization at 55 degreesC when glycerol is present. Activation involves displacement of the endogenous ATPase inhibitor subunit (epsilon subunit), and readdition of this subunit results in deactivation. In the deactivation process the ATPase inhibitor subunit can be replaced by other cationic proteins such as protamine, histones, or poly(lysine). Mg2+ and H+ also are effective deactivators. The fact that every positively charged substance tested deactivated the enzyme suggests that the inhibitor subunit is complexed with the enzyme at a site containing a surplus of negative charges. The activated enzyme is not labile, but it is salt labile, having a half-life of 2-3 min in 0.1 M KI at either 25 or 0 degrees C. The activated ATPase is also inhibited by aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD), and by the cross-linking agent dimethyl suberimidate. Evidence for polymorphism comes from finding that the properties of the unactivated enzyme (intrinsic ATPase) are different in many ways from the properties of activated ATPase. With respect to the coupling factor's ability to hydrolyze ATP, the data in this study suggest that there are at least four distinct functional allomorphs of this enzyme: (1) the latent enzyme, which has no kinetically measurable ATPase activity, (2) intrinsic ATPase, which is catalyzed by a small percentage of the molecular population that has been activated by some natural mechanism, (3) activated ATPase, which has properties different from those of intrinsic ATPase, and (4) aged activated ATPase, in which some of the properties (Km for substrate, sensitivity to deactivation by Mg2+ and H+) spontaneously change within 30 min.


Histidine decarboxylase of Lactobacillus 30a: function and reactivity of sulfhydryl groups. Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity.


Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast. The mechanism of action of yeast beta-hydroxy-beta-methylglutaryl-coenzyme A reductase has been investigated through kinetic studies on the oxidation of mevaldate by nicotinamide adeninine dinucleotide phosphate (NADP) in the presence of coenzyme A (CoA) and on the reduction of mevaldate by reduced NADP (NADPH) in the absence of presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, coenzyme A and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. These results suggest that CoA is an allosteric activator for the second reductive step and that it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the beta-hydroxy-beta-methylglutaryl-coenzymeA and the first NADPH. The binding of NADPH enhances the binding of the beta-hydroxy-beta-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. Thus, the replacement of NADP by NADPH after the first reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the second reductive step leave the enzyme before the release of the second NADP. This release of the last product is probably the rate-limiting step for the overall process.


Conformational changes in subfractions of calf thymus histone H1. This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.


Conformation of the extracellular polysaccharide of Xanthomonas campestris. The solution conformation of the extracellular polysaccharide of the bacterium Xanthomonas campestris is examined by optical rotation, viscometry, and potentiometric titration. Measurements of optical rotation vs. temperature for solutions of the polysaccharide at low ionic strength reveal a sharp transition to a denatured structure which is reversible if sufficient salt is present. The temperature Tm at the transition midpoint increases as log (Na+) or log (Ca2+). Viscosity-temperature profiles substantiate a structural change of the polysaccharide at Tm. The intrinsic viscosity of the native molecule at zero shear rate exceeds 5000 ml/g. This high figure is indicative of a stiff chain. The viscosity of the native molecule is relatively insensitive to salt, whereas the denatured molecule collapses if salt is present. Hydrogen-ion titration shows that the pKapp of the COO- groups of the polymer decreases from 3.2 in 0.01 M NaC1 to 2.6 in 0.2 M NaC1. All these data suggest that the native polysaccharide possesses ordered secondary structure stabilized by nonionic interactions outweighing the repulsion between adjacent COO- groups.


The reduction kinetics of chlorophyll aI as an indicator for proton uptake between the light reactions in chloroplasts. The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll aI (P-700) after far-red preillumination have been studied with high time resolution in spinach chloroplasts. 1. The kinetics of chlorophyll aI exhibits a pronounced lag phase of 2--3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll aI. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylkaloid membrane. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll aI reduction, is estimated as greater than 10(11) M-1s-1. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution. Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.


An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium. The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.