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Sanfilippo syndrome type C: deficiency of acetyl-CoA:alpha-glucosaminide N-acetyltransferase in skin fibroblasts.

Removal of N-sulfated glucosamine residues during degradation of heparan sulfate is accomplished by the sequential action of three enzymes. Action of sulfamidase results in the formation of alpha-glucosaminide residues. Removal of these groups requires conversion to alpha-N-acetylglucosaminide by the action of an acetyltransferase in the presence of acetyl-CoA, followed by hydrolysis by alpha-N-acetylglucosaminidase. In fibroblast homogenates from three patients with Sanfilippo syndrome type C (mucopolysaccharidosis III C), a biochemical variant of the Sanfilippo syndrome, complete deficiency of the acetyl-CoA:alpha-glucosaminide N-acetyltransferase activity was detected. Activities of all lysosomal hydrolases known so far to degrade mucopolysaccharides, including those of sulfamidase and alpha-N-acetylglucosaminidase, were in the range of controls. Acetyl-CoA:alpha-glucosaminide N-acetyltransferase activity was normal in fibroblasts of patients with other genetic mucopolysaccharidoses, including Sanfilippo syndrome A and B.

The position of regenerating cambia: auxin/sucrose ratio and the gradient induction hypothesis.

To account for the positions in which vascular cambia regenerate in wound callus, a gradient induction hypothesis was proposed in 1961 in terms of gradients in 'some factor as yet unknown'. It now seems likely that the gradient is based on morphogen diffusion between source and sink on opposite sides of existing cambia, with morphogen diffusing into the adjoining wound callus. It is specifically proposed that there are two morphogens, auxin diffusing centrifugally and sucrose diffusing centripetally. The cambium then regenerates along a path where the ratio of auxin to sucrose concentration is similar to that at the original cambium, and its orientation (as regards xylem and phloem formation) is determined by the direction of the gradient in this ratio. These proposals are supported by published evidence on auxin and sucrose concentration gradients across the cambium, and on their sources, movements, and known effects on vascular differentiation. Simulations of the proposed positional control system predict patterns of cambial regeneration and orientation corresponding to those observed in four different types of wound and graft.

[The determination of salivary pH by contact pH meter in individuals receiving psychotropic therapy. Study of pH on the tongue and at the orifice of Wharton's and Stenon's ducts (author's transl)].

The authors studied salivary pH at different sites in 172 patients receiving psychotropic therapy in hospital. For statistical purposes, the study was limited to individuals receiving a "moderate" dose of: --a benzodiazepine derivative --a neuroleptic and benzodiazepine --antidepressant, neuroleptic and benzodiazepine. Regardless of the type of psychotropic therapy and of the site of measurement, acidification of salivary pH which was statistically significant in comparison with values found in healthy subjects was noted. This acidification was particularly marked with regard to "lingual" pH. In addition, regardless of the site of measurement, a higher degree of acidification of salivary pH was seen in individuals receiving a combination of psychotropic agents. This acidification was particularly striking with regard to salivary pH in patients given a combination of antidepressant, neuroleptic and a benzodiazepine derivative. The cause of this acidification is not definitely known. A number of hypotheses may be put forward: decrease in salivary volume (Laudenbach, 6, and Vermeil, 7), excretion of acid metabolites by the salivary glands, effect of psychotropic agents upon the action itself of the salivary glands.

Arthritis and hepatitis.

The evidence relating four clinically distinct rheumatologic syndromes to infection by the hepatitis B virus is reviewed. Acute hepatitis B is not infrequently heralded by a prodromal rash and rheumatoidlike polyarthritis. Chronic active hepatitis B more rarely is associated with transient arthritis or arthralgias. Polyarteritis nodosa may be a manifestation of hepatitis B infection in as many as 40 percent of cases, and recently the syndrome of "essential" mixed cryoglobulinemia has also been linked to infection with this virus. The finding of immune complexes of varying composition, sometimes with the viral antigen or its antibody (or both) contained in both the serum and synovial fluid suggests that these four syndromes are clinical manifestations of immune complex disease resulting from hepatitis B infection.

ECG-changes in the fetal lamb during asphyxia in relation to beta-adrenoceptor stimulation and blockade.

Progressive changes in the S-T interval of the fetal electrocardiogram (FECG) were studied in 14 lamb fetuses, acutely exteriorized and subjected to graded hypoxia. The aims of the study were to investigate whether beta-adrenoceptor stimulation and hypoxia exerted additive or potentiating effects on the FECG and several cardiovascular parameters and whether the hypoxic changes of the FECG could be blocked by beta-adrenoceptor blocking agents. The FECG changes were studied in order to correlate them with cardiovascular function, as measured by heart rate, mean arterial pressure, end diastolic pressure, maximum dP/dt and combined cardiac output, estimated by the thermodilution method, as well as with blood gases, acid base status, blood lactate and glucose. Injections of small doses (0.02 to 0.4 microg kg-1 min-1) of isoprenaline induced the same pattern of changes in the FECG as we have previously recorded during hypoxia. By increasing the isoprenaline dose an increase in the duration of the FECG changes and amplitude of the T-wave changes was obtained. Propranolol was found to completely abolish the FECG changes induced by isoprenaline, as well as by mild hypoxia. During severe hypoxia the FECG changes could not be abolished by propranolol. Our previous findings indicated that the hypoxic changes could be regarded as a sign of myocardial glycolysis. Thus, the present finding that even small doses of isoprenaline given to the fetus, initiates the same pattern of FECG changes corroborate this hypothesis.

Age-dependent structural changes in human neuronal chromatin.

After partial digestion with micrococcal nuclease, DNA was extracted from nuclei of cerebral cortex neurons from young (23--36 y.) and old (78--85 y.) humans. The DNA fragments were subjected to gel electrophoresis, and their base-pair content determined. The nucleosomal DNA repeat length was found to increase from 170 (+/- 18) base-pairs in the young group to 199 (+/- 8) base-pairs in the old group. This increase of 29 base-pairs appears to be confined to the linker region of the nucleosomal DNA, since the core-DNA was always found to contain approx. 140 base-pairs. In addition, the amount of nuclear DNA digested by the micrococcal nuclease was observed to vary with age: after 30 min. of incubation at 37 degrees C hydrolysis of up to 80% of the nuclear DNA in the young but only up to 60% in the old neuronal nuclei was achieved. The age-dependent increase in chromosomal DNA repeat length is a direct proof of alterations in the basic chromatin structure with aging. It cannot be decided, however, whether the change in DNA digestibility is dependent on alterations of the chromatin basic structure, its superstructure, or both.

Clinical aspects of continuous tissular pH measurements of the newborn and the fetus.

The first results of continuous tissular pH readings in 58 neonates are presented. These results are compared with blood pH values and technical problems are discussed. The positive results made us proceed to sub-partu measurements. We finally give our opinion on the new ROCHE equipment and insist that it is not yet ready for routine use, but a promising tool for clinical research.

A study of the pH monitor in cats.

A study of the accuracy of the tpH electrode was performed in 10 cats. Hypoxia and acidosis were produced by ventilatory means and bicarbonate infusions were used to elevate the pH . Tissue pH correlated well with arterial (r = 0.9) within the physiological rates of change of pH. Prolonged tissue acidosis lead to eventual death. The correct and stable mechanical fixation of the pH electrode is a critical factor in assuring accurate results. The ROCHE tpH electrode accurately reflects arterial pH, and a correlation coefficient of at least 0.85 should be attainable in the human fetus.

Clinical and technical aspects of hemofiltration.

The removal of uremic substances in hemofiltration, in contrast to hemodialysis, is achieved by means of a convective transport across membranes of high porosity. Since 1974, more than 30 patients with chronic renal insufficiency have been treated with regular hemofiltration three times weekly for four to five hours each. After completing a pilot study, a controlled study to compare hemodialysis and hemofiltration was initiated during January, 1978. A normalization of blood pressure in patients with severe hypertension, and remarkable stability of the circulatory system, even after dehydration in patients who had hypotension in spite of fluid overload, could be demonstrated. Hemofiltration is preferred, especially in older patients with cardiovascular or cerebrovascular problems, because of its lower frequency of hypotensive episodes compared to dialysis. An important aim--the miniaturization of the artificial kidney--has not yet been achieved, however, because of the necessity for an extensive monitoring system for the exact proportioning of the sterile substitution fluid. First results in the application of a fluid regeneration system consising of a charcoal cartridge and a bioelectric cell, for degradation of urea, are presented.

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.

Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.

The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.

Ribavirin treatment in murine autoimmune disease. I. Therapeutic efficacy and effect on the immune response.

NZB/W F1 female mice were treated from 20 weeks of age with ribavirin (a broad spectrum antiviral drug), cyclophosphamide, or saline. Treatment with ribavirin (250 mg/kg twice weekly) prolonged survival from 9.8 to 18.5 months, reduced anti-DNA antibodies, and prevented proteinuria. Ability of ribavirin to prolong survival was dose related when given on a twice weekly schedule. However, daily ribavirin (25 mg/kg/day) was as effective as higher intermittent doses. Optimal ribavirin therapy was equal to cyclophosphamide treatment with regard to prolongation of survival. Ribavirin treatment did not significantly alter the body weight, hematocrit, WBC count, serum immunoglobulins, or Coombs reactivity. No alterations in either cellular or humoral immune responses were noted in NZB/W F1 or BALB/c mice treated for prolonged periods with ribavirin. The impressive therapeutic response to a broad spectrum antiviral agent seen in mice already manifesting immune complex nephritis provides a new therapeutic approach to the treatment of autoimmunity.

Reduction of methemerythrin by deoxymyoglobin: a protein-protein redox reaction not involving electron-transfer proteins.

The stoichiometry and kinetics of reaction of methemerythrin with the deoxy forms of myoglobin and hemoglobin have been examined at I = 0.2 M and 25 degrees C. One mole of methemerythrin (on the basis of the monomer unit containing two irons) reacts with 2 mol of deoxymyoglobin and with 0.5 mol of deoxyhemoglobin. All reactions are second order. Rate constants for reaction with deoxymyoglobin are 0.25 M-1s-1 (Phascolopsis gouldii) and 5.6 M-1s-1 (Themiste pyroides) at pH 6.3. There is little effect of raising the ionic strength to 1.35 M and only a small decrease in rate when the pH is adjusted to 8.2. The rate constant for reaction of deoxyhemoglobin with P. gouldii methemerythrin is approximately 0.1 M-1s-1 at pH 6.3. Metmyohemerythrin from T. pyroides reacts slightly slower than the octamer form (k = 2.0 M-1s-1 at pH 6.3 and 7.0). Oxymyoglobin is converted to metmyoglobin by methemerythrin. The electron-transfer path is discussed and a self-exchange rate constant for hemerythrin assessed as 10(-3) M-1s-1 on the basis of Marcus's theory.

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.

Sequential patterns of haemodynamic and metabolic changes in experimental hypovolaemic shock. I. Responses to acute haemorrhage.

Little is known about cardiorespiratory changes during the development of hypovolaemia. This study attempts to provide such information and compares the period of bleeding with that of established hypovolaemia. Eleven anaesthetized and ventilated greyhounds were bled and analyses of cardiopulmonary function made at fixed intervals both during and after haemorrhage. Six sequential patterns of cardiopulmonary and metabolic change were recognized. It was apparent that bleeding caused the first three phases of change, recovery from the effects of bleeding the next two and steady hypovolaemia the last. The event of bleeding is the main factor that elevates total peripheral resistance and reduces tissue perfusion with consequent lowering of oxygen consumption and alkalosis secondary to impaired carbon dioxide production; when bleeding ceases these changes partially reverse in a manner characteristic of that induced by the reinfusion of shed blood; and hypovolaemia per se has a relatively weak influence. These findings provide an explanation for disparities in previous published reports and have obvious clinical implications.

Unit activity of limbic system neurons: effects of morphine, diazepam and neuroleptic agents.

The effects of morphine, diazepam and three neuroleptic agents (chloropromazine, perphenazine and haloperiodol) on neuronal firing rats were studied in the limbic system of immobilized cats. Parietal craniotomy was carried out under 1.5--4.0% halothane. Extracellular potentials from single cells in the cingulate gyrus, septum and lateral hypothalamic areas were recorded using glass-coated, platinum-iridium microelectrodes. In general, intravenous adminstration of morphine sulfate augmented the spontaneous firing rates of most of the neurons studied. In contrast, diazepam produced a marked attenuation of both spontaneous and morphine augmented firing rates, whereas the neuroleptic agents had no significant or consistent effects on the morphine augmented firing rates of neurons in these limbic areas. These data indicate that the limbic system may play an important role in the behavioral excitement in cats induced by morphine administration and also the depressant effect of the tranquilizer diazepam. In contrast, the inability of the neuroleptic agents to antagonize the morphine augmented neuronal firing rates suggest these agents may act outside the limbic areas studied here.

The effects of bile on the gastric mucosa.

Pure rat bile, mixed with an equal volume of 43% ethanol or distilled water, was intubated into the stomachs of fasted rats. Control rats were intubated with saline solutions or received no intraluminal solutions into the stomachs. Following a 10 min period of exposure of the bile or saline solutions to the luminal surface of the gastric mucosa, samples of the stomachs were processed for routine scanning and transmission electron microscopy and light microscopy. Observations indicated that the bile solutions produced extensive damage to the luminal surface of the stomach. The most prominent alteration was evident as a massive cytolysis of the epithelial cells which line the gastric glands, pits, and luminal surface of the gastric mucosa. Loss of the epithelium revealed the underlying honey-combed structure of the lamina propria. In addition, haemorrhagic areas were often observed throughout the mucosa. The findings of this study indicated that bile mixed with water or ethanol was highly destructive to the gastric epithelium, even more destructive than a solution of ethanol and water; however, the basal lamina and connective tissue fibres of the lamina propria were apparently unaltered by the bile solutions. Furthermore, the presence of an intact lamina propria in many areas where there was not extensive haemorrhage, following exposure of these solutions to the gastric mucosa, suggested the existence of a scaffolding structure for the proper orientation of the reconstruction process of epithelium of the glands, pits, and luminal surface of the altered gastric mucosa.

The possible roles of membrane organization in the activity of androgen biosynthetic enzymes associated with normal or tumorous mouse Leydig cell microsomes.

The enzymes involved in conversion of pregnenolone to testosterone in Leydig cell tumors showed a wide distribution among smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and cytosol, while these enzymatic activities in normal testes were associated primarily with smooth endoplasmic reticulum. Progesterone, used as a substrate in the presence of an NADPH-generating system, was metabolized to androstenedione and finally to testosterone by microsomes from some strains of tumor which did not form testosterone from exogenous labeled androstenedione. Treatment of microsomal membranes from normal testes with 0.1 M Ca++ and Mg++ caused a marked decrease in 17 beta-dehydrogenase activity, measured as conversion of exogenous [3H]androstenedione to [3H]-testosterone, without serious effects on activities of 3 beta-ol-dehydrogenase or 17 alpha-hydroxylase. Studies of initial velocity kinetics showed that treatment with magnesium ion resulted in a marked reduction in affinity of androstenedione for 17 beta-dehydrogenase while the maximum velocity was the same as in untreated microsomes. Also, experiments using [14C]progesterone and [3H]androstenedione simultaneously as substrates demonstrated that treatment with Mg++ ion made it more difficult for exogenous [3H]androstenedione to reach the active site of 17 beta-ol-dehydrogenase than [14C]androstenedione formed in the microsomal membrane from [14C]progesterone. Microsomal proteins were more easily solubilized and 3 beta-ol-dehydrogenase was more severely influenced by Mg++ ion in tumor membranes than in normal microsomes.

Haemodynamic effects and pharmacokinetics of a new selective beta1-adrenoceptor agonist, prenalterol, and its interaction with metoprolol in man.

The haemodynamic effects of the selective beta1-adrenoceptor agonist prenalterol were studied in healthy subjects before and after therapeutic doses of the selective beta1-adrenoceptor blocker metoprolol. Plasma levels of the drugs were also determined in order to calculate certain pharmacokinetic variables. Intravenous infusion of prenalterol 0.13, 0.25 and 0.50 mg induced a dose-dependent decrease in total electromechanical systole (QA2) and pre-ejection period (PEP). The effect on left ventricular ejection time (LVET) was not significant. Increases in systolic blood pressure and heart rate were dose-dependent. Diastolic blood pressure did not change significantly. When metoprolol had been administered in a cumulative dose of 150 mg (mean maximal plasma level, 284 nmol/l) prenalterol had to be administered in doses that were twelve times higher than before the beta-blocker in order to induce the same haemodynamic effects. Prenalterol was rapidly distributed with an average half life of 8 min. This indicates that distribution equilibrium will be achieved within 30 min after intravenous administration. The overall elimination rate in the post-distributive phase corresponded to an average half life of 2.0 h.

Molecular properties of the adrenergic alpha-receptor. III - Origin of topographical dualism in the reaction of a receptor thiol with symmetrical and unsymmetrical polyamine disulfides.

Structure activity relationship studies previously led to the discovery of two distinct classes of tetramine-disulfides [(I) and (II)] each exhibiting optimum adrenergic alpha-blocking activity. The more potent one (I) was shown to uniquely discriminate between noradrenaline (NA) and adrenaline (A) binding sites. The unusual receptor topographical dualism toward (I) and (II) led to an investigation of the role of structural symmetry in their interaction with the receptor. To this end, a series of unsymmetrical disulfide analogs (x-z) where half of (I) was retained and the other half was made of unsubstituted N-(omega-aminoalkyl)cysteamines [as present in (II) and its homologs] were synthesized and evaluated as alpha-blockers. In addition, the role of the number of basic nitrogens on activity was also examined. It was found that the presence of four nitrogens is necessary for optimum activity. Moreover, it was discovered that optimum alpha-blocking potency is obtained when the respective halves of the two best prototypes (I) and (II) of symmetrical structures are fused [compound (XVI)]. This new unsymmetrical tetramine disulfide has a potency approaching that of (I) and its receptor saturation mechanism is similar. Moreover, it also shares with (I) the ability to discriminate between NA and A elicited responses. The effect of methylation of a single inner nitrogen on potency allowed the conclusion that each half of the unsymmetrical disulfide (XVI) respectively occupy half of the sites for (I) and half of those for (II). Accordingly, the previously observed topographical dualism toward (I) and (II) can best be accommodated by a model where two distinct sets of sites crossing each other over the same receptor thiol are involved. The possible significance of the anionic site multiplicity of the alpha-receptor is briefly discussed.

Amylase activity of Torulopsis ingeniosa Di Menna.

Torulopsis ingeniosa DI MENNA was found to possess an alpha-amylase strongly attached to the cell wall, its pH optimum being at 5.5, optimum temperature at 50 degrees C, highly sensitive to thermal inactivation. The enzyme was found to be induced by starch but the synthesis is not subject to a glucose effect.

Effect of growth rate and glucose concentration on the activity of the phosphoenolpyruvate phosphotransferase system in Streptococcus mutans Ingbritt grown in continuous culture.

Streptococcus mutans Ingbritt was grown anaerobically in a chemostat with a glucose limitation, as well as with an excess of glucose (amino acid limitation) at dilution rates (D) between 0.05 and 0.4 h(-1) (mean generation time = 12 to 1.5 h). The glucose-limited culture produced cells having 1.5- to 6.0-fold greater glycolytic activity than the cells from the glucose-excess culture. The preferred substrate for these cells was glucose, with the glycolytic rate for sucrose being only slightly lower; the rate for fructose was half that of glucose. The glycolytic rate of the glucose-limited cells was maximum at D = 0.1 h(-1), with a decline in rate as the growth rate approached D = 0.4 h(-1). A comparison of the activity of phosphoenolpyruvate phosphotransferase system (PTS) in the two types of cells showed that the glucose-limited cells had 1.7- to 5.6-fold greater PTS activity for the three sugars than the glucose-excess-grown cells. Whereas little difference was seen between the three sugars with the latter cells, the glucose-PTS had the greatest activity with glucose-limited cells, with the maximum in cells grown at D = 0.1 h(-1). Comparison of the rate of sugar uptake in the chemostat with the rate of PTS transport activity in the cells at each growth rate demonstrated that only under conditions of slow growth with a glucose limitation was the PTS system capable of supporting growth on glucose. Furthermore, PTS activity in cells grown with an excess of glucose was insignificant when compared with glucose uptake during growth in the chemostat. This evidence supports the observation that S. mutans possesses at least one other system, in addition to the PTS, for the transport of glucose into the cell. The organism was, however, devoid of glucose-proton symport transport activity.

Recent advances in diagnosis and management of galactorrhea.

Using prolactin suppression (L-dopa) and provocative (thorazine) tests along with the clomid test the intactness of hypothalamico-pituitary axis was assessed in a group of eight patients with galactorrhea syndrome. Pituitary neoplasm was suspected in three cases on the basis of elevated plasma prolactin and its abnormal response to L-dopa, thorazine, and clomid tests. Trans-sphenoidal exploration of the pituitary gland in these three cases confirmed the diagnosis. In these cases, however, the commonly used parameters for evaluation of the pituitary gland (skull X ray, visual field measurement, echoencephalogram, etc.) failed to reveal the presence of the pituitary lesions. In two of the three cases, normal menses recurred following extirpation of the tumors and conception was achieved successfully. In the remaining patient the operation failed to remove the entire pituitary lesion and it was necessary to irradiate the pituitary gland as the patient continued to have persistent galactorrhea and hyperprolactinemia. The importance of the prolactin assay, and its response to stimulation and suppression tests for proper evaluation of galactorrhea was fully discussed.

The separation of peptide hormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers--structural implications.

Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, [2-D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, and [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the muBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.

Glutamine synthetase regulation, adenylylation state, and strain specificity analyzed by polyacrylamide gel electrophoresis.

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GS(E)) and Klebsiella aerogenes (GS(K)). In gels containing sodium dodecyl sulfate (SDS), we found that GS(K) had a mobility which differed significantly from that of GS(E). In addition, for both GS(K) and GS(E), adenylylated subunits (GS(K)-adenosine 5'-monophosphate [AMP] and GS(E)-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GS(K)-AMP < GS(K) < GS(E)-AMP < GS(E). We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GS(K), and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GS(K) or GS(K)-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele.

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.

Carbodiimide-binding protein of H+-translocating ATPase and inhibition of H+ conduction by dicyclohexylcarbodiimide.

H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified F0 from a thermophilic bacterium PS3 (TF0), the following results were obtained. 1. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5 times 10(3) M(-1)-min(-1). 2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+. 3. By extracting TF0 with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6,000. 4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6,500. 5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.

A trypsin-like oviducal proteinase involved in Bufo arenarum fertilization.

When the jelly-less eggs removed from the most cephalic region of the oviduct (pars recta) of the toad Bufo arenarum were inseminated at a high sperm concentration, high frequencies of fertilization were obtained. On the other hand, control eggs removed from the pleuroperitoneal cavity (coelomic eggs)) were neither fertilized upon insemination under identical conditions, nor with the water extract of the jelly. Under these inseminating conditions, however, a high frequency of fertilization was obtained when coelomic eggs were preincubated in the presence of the fluid secreted by the epithelium of the pars recta or of an extract prepared from pars recta homogenate. Experimental evidence is presented showing that the component responsible for this effect acts on the vitelline envelope of the egg, increasing its susceptibility to sperm lysin. It is probable, therefore, that it induces successful fertilization of coelomic eggs by making the vitelline envelope more easily penetrable by sperm. The active factor was partially purified by Sephadex chromatography. The product obtained was of high activity and, as judged by its inhibition with soybean trypsin inhibitor and lima-bean trypsin inhibitor, it is likely to be a trypsin-like enzyme. The molecular weight of the factor was estimated to be 47000 by Sephadex chromatography. Secretion of the pars recta factor is hypophysis-dependent and its activity is not influenced by pH within the range testes (6.0--8.4).

Solubility of doxycycline in aqueous solution.

The solubility of doxycyline monohydrate and doxycycline hydrochloride dihydrate was investigated in aqueous solution. The hydrochloride dihydrate salt was isolated and identified from solutions initially containing doxycycline hyclate in water. The pKa' = 3.09 (mu = 0.1 and 25 degrees) for protonation of doxycycline was determined spectrophotometrically. The pH-solubility profiles were determined for doxycycline monohydrate in water and in 1.0 M NaNO3-HNO3 and NaCl-HCl. The pH-solubility profile at 25 degrees for doxycycline in aqueous hydrochloric acid without added salt reached a sharp maximum fo 50 mg/ml at pH 2.16. Added chloride ion strongly suppressed the solubility of the hydrochloride dihydrate salt. The apparent solubility product was not constant but decreased as the concentration of added salt increased. A theoretical model was developed involving dimerization of doxycycline and applied to the experimental data. The dimerization constant, Kd = 24 M-1, and true solubility product, K0sp = 1.8 X 10(-3) M2, were calculated. The effect of concentration on NMR and visible spectra indicated that dimerization resulted from intermolecular hydrogen bonding of the phenolic beta-diketone portion of the molecule.

The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma.

Epididymal spermatozoa from bull, rabbit and ram were incubated in homologous epididymal plasma or seminal plasma in a buffered saline-based medium with or without serum albumin. The spermatozoa were either diluted directly into the medium or were washed first. No effect of washing was observed on the subsequent reaction of the cells to the different media. A considerable proportion of the populations of epididymal spermatozoa survived (i.e. continued to exhibit motility) for up to 22 h at 30 degrees C in the simple saline-based medium. Initially epididymal plasma had a slight stimulatory effect on sperm motility in ram and bull but it had no effect on sperm survival in any of the 3 species. Seminal plasma stimulated motility markedly in ram initially, but in all 3 species seminal plasma was detrimental to survival: in ram even a 15-min exposure to the fluid reduced survival. Serum albumin also stimulated motility; it delayed, but did not prevent, the detrimental effect of seminal plasma, although it had no effect itself on survival. The effects of epididymal plasma, seminal plasma and serum albumin on surface properties of epididymal spermatozoa, i.e. agglutination, sticking-to-glass and eosinophilia, were also noted. These varied between species and there was no correlation between these effects and the effects on motility and survival.

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis.

The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes. The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O2 and H2O2, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O2- and H2O2 by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H2O2 is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H2O2 degrading enzymes in guinea pig cells. The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported. It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H2O2 degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.

Substance P: characteristics of binding to synaptic vesicles of rat brain.

1. The binding of substance P (SP) to synaptic vesicles from rat brain was studied by use of the 125I-Tyr8-analogue of SP. 2. The pH dependence of the binding of both peptides to the lipid extractable fraction of synaptic vesicles was shown to be comparable. 3. The binding of 125I-Tyr8-SP shows a rate constant of association (k1 = 6.6 x 10(6) M-1 S-1), a rate constant of dissociation (k-1 = 6.4 x 10(-4) S-1) and gives a KD of 1 x 10(-10) M. Kd derived from equilibrium studies was 3.2 x 10(-10) M. 4. The binding of 125I-Tyr8-SP to lipids of synaptic vesicles was shown to be reversible, saturable and highly specific. 5. The kinetic data suggest one population of binding sites with a maximal number of 0.8 pmol per mg protein of the synaptic vesicle preparation. 6. Unlabeled SP and the (2--11)-, (3--11)- and (4--11)-analogues of SP inhibit the binding of 125I-Tyr8-SP in a decreasing order in a competitive way when added in excess. Tyr8-SP and eledoisin did not interfere with the binding of 125I-Tyr8-SP whereas uperolein and neurotensin caused a partial inhibition. Physalaemin and D-Ala2-D-Met5-enkephalin enhance the binding of 125I-Tyr8-SP in a cooperative way.

Antibodies distinguishing between intact and alkali-hydrolyzed 7-methylguanosine.

Antibodies specific for intact 7-methylguanosine (m7G) were induced in rabbits and mice by immunization with nucleoside-BSA or nucleoside-hemocyanin conjugates. Since m7G undergoes alkali-catalyzed hydrolytic fission of the purine ring, modifications were made in the procedure for conjugation of m7G to proteins. After periodate oxidation, m7G was incubated with protein at pH 9.1 at 4 degrees C for one hour during which the nucleoside was found to be stable. Reduction of the Schiff base was done with t-butylamine borane for 30 minutes, and the conjugated protein was isolated quickly by gel filtration at pH 7.2. Both rabbits and mice produced antibodies that readily distinguished between the intact and hydrolyzed m7G. Antibody specificity depended largely on the presence of an intact 7-substituted imidazole ring and some cross-reaction occurred with 7-methylinosine. A weaker reaction occurred with ribothymidine and thymidine. Mouse antibodies induced by m7G-hemocyanin showed the highest specificity. They also recognized m7G in the isolated mRNA cap structure m7G(5')ppp(5')A.

beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling.

The beta-adrenergic agonist L-isoproterenol stimulated the enzymic synthesis of phosphatidyl-N-monomethylethanolamine and phosphatidylcholine in rat reticulocyte ghosts containing the methyl donor S-adenosyl-L-methionine. The stimulation was stereospecific, dose-dependent, and inhibited by the beta-adrenergic agonist propranolol. The addition of GTP inside the resealed ghosts shifted the dose-response of phospholipid methylation by L-isoproterenol to the left by 2 orders of magnitude. Direct stimulation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with sodium fluoride or cholera toxin did not increase the methylation of phospholipids. At a concentration of S-adenosyl-L-methionine that stimulates synthesis of phosphatidyl-N-monomethylethanolamine, the activity of isoproterenol-sensitive adenylate cyclase was increased 2-fold without changes in the basal activity of adenylate cyclase and the number of beta-adrenergic receptors. The increase of phospholipid methylation by L-isoproterenol decreased membrane viscosity and increased translocation of methylated lipids. These findings indicate that enhancement of phospholipid methylation by L-isoproterenol decreases membrane microviscosity and thus increases lateral movement of the beta-adrenergic receptors and coupling with adenylate cyclase.

The correlation between discharge times of neighbouring neurons in isolated cerebral cortex.

The purpose of the experiments was to find out whether neighbouring neurons in chronic preparations of neurally isolated cerebral cortex are more likely to fire synchronously than are similar neurons in the intact brain. Chronically implanted extracellular microelectrodes were used to obtain simultaneous records of the spontaneous discharges of neighbouring neurons in the suprasylvian gyrus of the unanesthetized, unrestrained cat. We have examined multi-unit records obtained from neurons in islands of neurally isolated cortex; these records have been compared with similar records from neurons in the same cortical region of the intact brains of control animals. In isolated cortex, neighbouring neurons showed a tendency to discharge in near synchrony. In contrast, there was a random temporal relation between the firing times of adjacent nerve cells of intact cortex, provided the cat was awake. These results, taken together with the relevant observations of other workers, may indicate the manner in which biologically important information is transmitted within the mammalian brain.

Individual variations in the effects of flurazepam, clorazepate, L-dopa and thyrotropin-releasing hormone on REM sleep in man.

The comporative effects of flurazepam, clorazepate, L-dopa, and thyrotropin-releasing hormone (TRH) on REM sleep were investigated in normal, healthy adults. A single dose of each drug was given orally to the subjects 30 min before bedtime. A dose of 30 mg flurazepam significantly decreased REM sleep-time when compared to the mean baseline record. No change was noted in REM sleep-time on the clorazepate (15 mg) night, L-dopa (1000 mg) night, or TRH (2 mg) night, when compared to the mean baseline record. Because large individual variations were found in REM sleep time on each drug night, and in percentage increase in REM sleep following partial differential REM deprivation (PDRD), correlation was investigated between them. The percentage decrease in REM sleep during flurazepam was found to have a significant negative correlation with the percentage increase in REM sleep after PDRD in individual subjects. Although there was no significant change in REM sleep on TRH night when compared to the mean baseline record, a similar significant negative correlation was noted. On the L-dopa night, there was a tendency toward a negative correlation between them. No significant correlation was noted on the clorazepate night.

[Antimicrobial resistance and serotypes of Streptococcus pneumoniae in Switzerland].

One hundred and eighty strains of Streptococcus pneumoniae were isolated from various materials at the Institute of Medical Microbiology in Zurich between March and October 1978. Examination of these isolates for resistance to 6 standard antimicrobials revealed that 17 (9.4%) strains were resistant to the tetracyclines, 2 strains to sulphamethoxazole/trimethoprim and 1 strain to both tetracycline and chloramphenicol. All strains were highly sensitive to benzylpenicillin, cephalothin and erythromycin. Serotyping of the pneumococci was done by the capsular quellung reaction. The prevalent serotypes in the entire collection of strains were Nos. 6, 23, 19, 3, 9, 14 and 7. The prevalent serotypes of strains isolated from blood, cerebrospinal fluid, pus, and bronchial secretions were Nos. 4, 8, 7, 14, 9, 3, 19 and 6. Vaccination with PneumovaxTM, a vaccine containing 14 capsular antigens, should provide protection against 75% of pneumococci identified as true pathogens in this study. Pneumococci are rather rarely isolated in our laboratories. For this reason, and because the strains isolated are still sensitive to antibiotics in conventional use, mass vaccination of the population in Switzerland is not recommended. Vaccination should be carried out in certain high risk groups of patients.

[Genetically conditioned variability of metabolic profile parameters in dairy cows].

Individual heritability and differences in the concentration of the chemical components of the blood were studied in the dairy cows of the Slovak Spotted breed. The experiment was performed with 166 cows. The set comprised six groups of half-sisters from three stocks. The differences among the cows were statistically significant (alpha = 0.01) in the majority of the parameters studied: haematocrit, haemoglobin, pH, PO2, oxygen saturation of the blood; plasma potassium, phosphorus, magnesium, calcium, total protein, urea, glucose, alkaline phosphatase, and esterified fatty acids. The coefficients of repeatibility for the mentioned parameters ranged from 0.19 to 0.75. The heritability coefficients were calculated for the parameters in which the inter-group differences were significant: total protein (0.62), magnesium (0.57), potassium (0.51), urea (0.49), glucose (0.45), phosphorus (0.43), calcium (0.39), haematocrit (0.37), haemoglobin (0.35), pO2 (0.29). The results suggest that some of the parameters under study are under certain genetic control.

Bone marrow transplantation.

Bone marrow transplantation can be considered in any disease state resulting in the malfunction or absence of part or all bone marrow elements. Diseases such as aplastic anemia, leukemia, and immunodeficiency disease are being treated with bone marrow transplantation. As with any organ transplant, graft rejection is a possibility. In bone marrow transplantation, there is the additional, unique problem of graft versus host disease. In order to prevent or minimize graft rejection, the immunocompetence of the recipient and the degree of disparity between donor and recipient at the major histocompatibility complex (MHC) loci are considered. The results of bone marrow transplantation are variable, and the mortality rate is still relatively high. However, progress is being made, and in many instances, normal bone marrow function can be restored in patients with whom other treatment has failed.

Impaired renal tubular potassium secretion in sickle cell disease.

We examined renal tubular function in six patients with sickle cell hemoglobin. All had normal inulin and para-aminohippurate clearances and impaired urinary concentrating and acidifying abilities. After intravenous potassium chloride administration, maximum excretion of potassium (U,V) was significantly lower in sickle cell patients than in control subjects, and the percentage of potassium load excreted in 5 h was markedly reduced. Urinary potassium excretion after sodium sulfate infusion was also markedly reduced in sickle cell patients compared to control subjects. After 40 mg of oral furosemide, U,V was also diminished in sickle cell patients. Plasma aldosterone response to ACTH and intravenous potassium was similar to that of control subjects. Plasma renin activity increased normally after volume contraction. We conclude that sickle cell patients have a defect in their ability to excrete an acute potassium load that cannot be attributed to abnormal renin or aldosterone secretion. Overall potassium homeostasis is maintained by extrarenal mechanisms during acute potassium loading.

Acetoacetate metabolism in rat brain. Development of acetoacetyl-coenzyme A deacylase and 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

1. Data are provided that indicate that the rat brain acetoacetyl-CoA deacylase is almost exclusively mitochondrial. Developmental studies show that this enzyme more than doubles its activity during suckling (0--21 days) and then maintains this activity in adults (approx. 1.1 units/g wet wt.). 2. Kinetic studies (on the acetoacetyl-CoA deacylase) in a purified brain mitochondrial preparation give a Vmax. of 47 nmol/min per mg of protein, and a Km for acetoacetyl-CoA of 5.2 micron and are compatible with substrate inhibition by acetoacetyl-CoA above concentrations of 47 micron. 3. The total brain 3-hydroxy-3-methyl-glutaryl-CoA synthase remains constant in the developing and adult rat brain (approx. 1.2 units/g wet wt.). This enzyme is located in both the mitochondrial and cytosolic fractions. During suckling (0--21 days) the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase represents approx. one-third of the total, but this increases markedly to about 60% of the total in the adult. The cytosolic enzyme correspondingly falls to approx. 40% of the total. 4. The role of the acetoacetyl-CoA deacylase in providing cytosolic acetoacetate for biosynthetic activities in the developing brain is discussed.

Acetyl-coenzyme A deacylase activity in liver is not an artifact. Subcellular distribution and substrate specificity of acetyl-coenzyme A deacylase activities in rat liver.

Whole liver and isolated liver mitochondria are able to release free acetate, especially under conditions of increased fatty acid oxidation. In the present paper it is shown that rat liver contains acetyl-CoA deacylase (EC 3.1.2.1) activity (0.72mumol/min per g wet wt. of liver at 30 degrees C and 0.5mm-acetyl-CoA). At 0.5mm-acetyl-CoA 73% of total enzyme activity was found in the mitochondria, 8% in the lysosomal fraction and 19% in the postmicrosomal supernatant. Mitochondrial subfractionation shows that mitochondrial acetyl-CoA deacylase activity is restricted to the matrix space. Mitochondrial acetyl-CoA deacylase showed almost no activity with either butyryl- or hexanoyl-CoA. Acetyl-CoA hydrolase activity from purified rat liver lysosomes exhibited a very low affinity for acetyl-CoA (apparent K(m)>15mm compared with an apparent K(m) value of 0.5mm for the mitochondrial enzyme) and reacted at about the same rate with acetyl-, n-butyryl- and hexanoyl-CoA. We could not confirm the findings of Costa & Snoswell [(1975) Biochem. J.152, 167-172] according to which mitochondrial acetyl-CoA deacylase was considered to be an artifact resulting from the combined actions of acetyl-CoA-l-carnitine acetyltransferase (EC 2.3.1.7) and acetylcarnitine hydrolase. The results are in line with the concept that free acetate released by the liver under physiological conditions stems from the intramitochondrial deacylation of acetyl-CoA.

Fluorescence studies of native and modified neurophysins. Effects of peptides and pH.

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by Förster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.

Isolation and partial characterization of intermediate filament protein (skeletin) from cow heart Purkinje fibres.

The intermediate filament protein skeletin from cow heart Purkinje fibres was purified to homogeneity by a selective extraction procedure and gel chromatography in the presence of sodium dodecyl sulphate. Monospecific antibodies were obtained by immunisation of rabbits with the sodium dodecyl sulphate-skeletin complex, and rocket electrophoresis made it possible to quantify the concentration of protein. The skeletin monomer has a molecular weight of 55 000. Amino acid analysis revealed that skeletin has a high content of glutamic acid, aspartic acid, alanine and leucine, together constituting more than 50% of the molecule. The isoelectric point is determined as 6.35. Skeletin is insoluble at pH 4--6 in the absence of detergent and shows increasing solubility at higher and lower pH. The biochemical characteristics are discussed in relation to the cytoskeletal function of the filaments. Comparison with intermediate-sized filament protein of other tissues show certain important similarities suggesting that the filaments may share a common evolutionary ancestry.

Long-term effects of preganglionic nerve stimulation on tyrosine hydroxylase activity in the rat superior cervical ganglion.

The effects of increased synaptic stimulation of sympathetic neurons on the tyrosine hydroxylase activity of these cells were studied. Seventy-two hours after unilateral stimulation of the preganglionic cervical sympathetic trunk at 10 Hz for 30 min tyrosine hydroxylase activity was 32% higher in stimulated than in control superior cervical ganglia. Stimulation at 10 Hz for only 10 min increased enzyme activity by 25% when measured 72 h later, while stimulation for 60 min increased activity by 73%. No further change in enzyme activity was found after 90 min of stimulation although electrophysiological recordings from the ganglion demonstrated that synaptic transmission was maintained throughout the period of stimulation. Ganglionic neurons also follow high frequency trains of stimuli when they are interrupted by silent periods. Stimulation with 40 Hz trains (250 msec on/500 msec off) for 30 min and 90 min produced a 50% and a 92% increase in tyrosine hydroxylase activity, respectively. Stimulation of ganglia with the same number of pulses administered either continuously or in trains produced the same size increase in enzyme activity. The relationship between preganglionic nerve activity and tyrosine hydroxylase activity may represent an adaption of sympathetic neurons to situations requiring increased transmitter release.

Reactions of nitrosobenzene with reduced glutathione.

Nitrosobenzene (NOB) formed acid labile conjugates with reduced glutathione (GSH) and hemoglobin within red cells. In vitro, NOB rapidly reacted with GSH with formation of phenylhydroxylamine (PH), oxidized glutathione (GSSG), and a water-soluble compound identified as glutathionesulfinanilide (GSO-AN). Free aniline (AN), aminophenols and azoxybenzene were not detected. The proportion of PH formed increased with increasing GSH concentration and at higher pH values. Spectroscopic analysis revealed the formation of a labile adduct following a second order reaction (K = 5 x 10(3) M-1 . sec-1 at pH 7.4 and 37 degrees). This reaction was reversible because nearly all NOB could be extracted with ether from the labile intermediate. On the other hand, the labile intermediate was transformed into GSO-AN (with increasing rate at lower pH values) or it was cleaved by GSH with formation of GSSG and PH. Intermediate formation of NOB and thiol radicals was ruled out by analysis of the equilibrium data. A tentative scheme is presented for the proposed reaction mechanism.

Synthesis and activation of asparagine in asparagine auxotrophs of Saccharomyces cerevisiae.

L-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms. Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA- and asnB-. Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutationalone. This, and Jones' report [J. Bacteriol. 134, 200-207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in sigma 1278b wild type) is cancelled by casnI- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant.

Partial purification of a toxin found in hamsters with antibiotic-associated colitis. Reversible binding of the toxin by cholestyramine.

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.

Limited proteolysis of glutamine synthetase is inhibited by glutamate and by feedback inhibitors.

Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.

Evaluation of blood culture procedures in a pediatric hospital.

To determine optimal clinical laboratory techniques for detecting pediatric bacteremia, we studied 7,768 consecutive blood cultures in a 1-year period. Blood was inoculated into one vented 50-ml bottle of brucella broth with 0.05% sodium polyanetholsulfonate and one unvented 50-ml bottle of Columbia broth with 0.05% sodium polyanetholsulfonate and 0.05% cysteine. Bottles were visually examined for growth on days 1 through 7 and blindly subcultured aerobically and anaerobically on days 1, 2, and 7. There were 724 (9.3%) positive cultures, and 484 (6.2%) were clinically significant. The most frequent isolates from bacteremic patients were Haemophilus influenzae (24%) and Streptococcus pneumoniae (17%). Growth was noted in only one bottle in 25% of clinically significant isolates. Bottles inoculated with greater than or equal to 1 ml of blood became positive earlier than bottles inoculated with less than 1 ml. After 1 day of incubation, 48% of the clinically significant cultures showed growth on visual examination, whereas 85% showed growth on subculture. Only 19% of Haemophilus isolates were detected visually on day 1, whereas 88% were recovered on subculture. By day 7, 3.5% of all isolates (including 18% of pneumococcal isolates and 1% of Haemophilus isolates) could no longer be recovered on subculture. We conclude that a two-bottle blood culture system and blind subculture within 24 h will optimize detection of pediatric bacteremia.

Dopamine during alpha- or beta-adrenergic blockade in man. Hormonal, metabolic, and cardiovascular effects.

We studied the contribution of alpha- and beta-adrenergic receptor activation to the cardiovascular, metabolic, and hormonal effects of dopamine. At a concentration of 1.5 mug/kg.min, the infusion of dopamine in 12 normal volunteers was associated with a transient but significant rise in pulse rate, which was prevented by propranolol. Venous plasma glucose did not change throughout the experiments, and a mild increase in plasma free fatty acid levels observed during the administration of dopamine alone was antagonized by propranolol. In contrast, neither the beta-adrenergic blocker, propranolol, nor the alpha-adrenergic blocker, phentolamine, was effective in inhibiting the dopamine-induced rise in plasma glucagon (from 82+/-9 to 128+/-14 pg/ml; P < 0.005) and serum insulin (from 7.5+/-1 to 13+/-1.5 muU/ml; P < 0.005) or its suppression of plasma prolactin (from 8.5+/-1 to 5.2+/-0.8 ng/ml; P < 0.001). Although serum growth hormone levels did not change during the infusion of dopamine alone, an obvious rise occurred in three subjects during the combined infusion of propranolol and dopamine. Whereas some metabolic and cardiovascular effects of dopamine are mediated through adrenergic mechanisms, these observations indicate that this is not the case for the effects of this catecholamine on glucagon, insulin, and prolactin secretion, and thus provide further support for the theory of a specific dopaminergic sensitivity of these hormonal systems in man.

[Obstetrical anaesthesia. Their place in the conduct of labour (author's transl)].

After having made an historical study of the principal works coming from the School in Toulouse concerning obstetrical anaesthesia, one can make an account of the criteria for fetal risk. These have been established by comparing with a standard that determines the physical and biological parameters found in normal labour. The fetal risk has been studied for four types of anaesthesia progressively. These are : general anaesthesia with Pentothal, general anaesthesia with Gamma OH, neuroleptanalgesia and finally epidural anaesthesia. By comparison with a normal standard, the evolution of the parameters concerning uterine contraction, the changes in the fetal heart rate the, acid-base balance of the fetal blood and how it changes, and the Apgar score have been studied for each type of anaesthesia. The same has been done for maternal risk. After having completed this study we have come to the conclusion that there is no single anaesthetic that should particularly be recommended, and that for every phase of labour : before labour starts and after labour has been confirmed, and at the end of the first stage of labour, a different form of anaesthesia may well be indicated. Above all, the skill of the anaesthetist and of the obstetrician influence the efficiency of the anaesthetic.

Studies of experimental cervical spinal cord transection. Part III: Effects of acute cervical spinal cord transection on cerebral blood flow.

Regional cerebral blood flow (CBF) was measured by the microsphere technique in anesthetized, mechanically ventilated dogs before and after cervical laminectomy in four (control group), or cervical laminectomy followed by cervical cord transection (CCT) at the C-6 level in six (experimental group). No significant differences in arterial pH, pO2 or pCO2 were observed between control and experimental dogs. Baseline values for mean arterial pressure (MAP) were also similar in the two groups, but MAP fell in all experimental dogs after CCT (p less than 0.025). At 120 minutes after CCT, three of the six dogs had an MAP greater than 60 torr (66 +/- 4 torr), and in three the MAP was greater than 50 torr (45 +/- 3 torr). Regional CBF in cortical gray matter, white matter, and medulla did not change significantly after CCT in dogs with MAP greater than 60 torr. The CBF fell significantly at 120 minutes after CCT in all regions sampled in the dogs with MAP less than 50 torr (p less than 0.025). At 30 and 120 minutes after CCT, cerebellar blood flow fell significantly in all experimental animals (p less than 0.05). These findings indicate that, despite hypotension and sympathetic denervation of cerebral vessels, CBF in cortical gray matter, white matter, and medulla is maintained at normal levels after CCT by autoregulation as long as MAP exceeds 60 torr. Decreased cerebellar blood flow in the experimental group suggests redistribution of CBF after CCT with relative preservation of flow to gray matter, white matter, and medulla. Reduced CBF in the acutely cord-injured patient with significant hypotension (MAP less than 60 torr) may stimulate or complicate coexistent head injury.

The gastrointestinal absorption of paracetamol in the rat.

The absorption of [3H]paracetamol by rat small intestine, colon and stomach was studied in vivo and in vitro. Small intestinal in vivo studies, using a wide range of drug concentrations, showed that absorption was efficient and uniform throughout the small bowel, no site showing preferential absorption. Double reciprocal and direct plots indicated first order kinetics. The pattern was not observed when uptake was occurring from high concentrations of paracetamol in suspension. Gastric and colonic in vivo studies showed that there was appreciable absorption of [3H]paracetamol from these sites. In vitro studies using everted intestinal sacs showed no effect on paracetamol transfer when the incubation temperature was lowered to 10 degrees C or when iodoacetate (5 X 10(-2)M) and 2.4 dinitrophenol (5 X 10(-4)M) was added to the incubation medium. There was, however, a significant reduction in transfer of paracetamol against a concentration gradient of 10:1 applied across the mucosa. These data suggest that the uptake of paracetamol is by a passive transport process and confirm the efficiency of paracetamol absorption observed indirectly by others.

Activation of liver guanylate cyclase by paraquat: possible role of superoxide anion.

Paraquat, a herbicide which is known to increase intracellular levels of superoxide anion (O2-), stimulated guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.] activity. This stimulation by paraquat was seen at concentrations as low as 0.005 mM. The activation of guanylate cyclase by paraquat was not blocked by KCN, an inhibitor of superoxide dismutase [EC 1.15.1.1.], suggesting that the activation process probably does not involve superoxide dismutase which converts superoxide anion to hydrogen peroxide and ultimately to hydroxyl radical. Catalase [EC 1.11.1.6.] did not block the paraquat activation of guanylate cyclase indicating that hydrogen peroxide was probably not involved in the activation process. Butylated hydroxytoluene, a hydroxyl radical scavenger, also had no effect on the paraquat activation of guanylate cyclase activity. Superoxide dismutase inhibited the paraquat activation of guanylate cyclase. Thus, it would appear that superoxide ion itself can activate guanylate cyclase circumventing any requirement for hydroxyl radical formation.

Intergeneric and intrageneric inhibition between strains of Propionibacterium acnes and micrococcaceae, particularly Staphylococcus epidermidis, isolated from normal skin and acne lesions.

Two hundred and forty-one strains or resident skin bacteria comprising 93 isolates of Propionob acterium acnes and 148 of Micrococcaceae derived from 36 acne patients and 8 control subjects were screened for their ability to inhibit 32 indicator strains, including 20 strains of P. acnes and 12 strains of Staphylococcus epidermidis derived from patients with all grades of acne and from normal skin. Fifty-three strains (22%) showed some activity against at least one indicator strain. Both broad- and narrow-spectrum inhibition was detected. Inhibitory isolates of P. acnes outnumbered inhibitory Micrococcaceae by four to one. There was a low frequency of inhibition of S. epidermidis by Micrococcaceae (2.7%) and by P. acnes (1.1%) and a higher frequency of inhibition of P. acnes by Micrococcaceae (9.5%) and by P. acnes (40.8%). Furthermore, 81.8% of the subjects sampled possessed strains inhibitory to P. acnes. The significance of this finding is, as yet, unknown. No difference in the prevalence of active strains in normal (20%) and acne (22.5%) skin was detected. These findings suggest that the possession of inhibitory strains and conversely the possession of sensitive strains does not predispose to acne.

[Necrotizing angiitis of small vessels. A clinical study of 25 patients with skin biopsy (author's transl)].

Necrotizing angiitis or vasculitis exhibits a wide clinical spectrum characterized by many different cutaneous manifestations. Diagnosis must be confirmed by histopathology. We studied in retrospect 25 patients whose conditions had been diagnosed by skin biopsy. Histologic examination revealed infiltration by polynuclear cells and fibrinoid necrosis of the walls of the blood vessels in the skin. The great variety of clinical manifestations and etiologies stands out in a review of the records of these patients. Necrotizing angiitis has been found associated with mixed cryoglobulinemia; administration of drugs, milliary tuberculosis, bacterial meningitis, rickettsiosis, staphylococcal sepsis, pharyngotonsillitis, and rheumatoid arthritis. Necrotizing angiitis is a group of diseases with a great variety of clinical manifestations, ranging from benign to fatal. The various entities described to date have been more like different clinical forms of the same disease that distinct conditions. In cases of necrotizing angiitis caused by basically immunological mechanisms, the walls of the blood vessels may be impaired in varying diffuse degrees. The prognosis of the disease depends on the intensity of the inflammation and its repercussions on the parenchymas of different organs. The kidney is the most susceptible organ in this case. Treatment should be directed toward the avoidance of predisposing and etiologic factors, detection of the immunological reaction, requiring careful and individual attention in every case.

Lergotrile in Parkinson disease: further studies.

Lergotrile was administered to 53 patients with advanced Parkinson disease (PD), who had increasing disability despite optimal treatment with levodopa/carbidopa (Sinemet). Thirty-nine patients who could tolerate at least 20 mg per day lergotrile (thus considered "adequately treated") had significant descreases in rigidity, tremor, bradykinesia, gait disturbance, and total score without increased involuntary movements. Twenty-one of these 39 patients improved by at least one stage. Among the 39 patients, 23 had "on-off" effects, and in 13 of these the "on-off" effects decreased on lergotrile. The mean daily dose of lergotrile in adequately treated patients was 49 mg, permitting a 10 percent reduction in the dose of levodopa. Lergotrile was discontinued in 33 of the 53 patients because of adverse effects, including hepatotoxicity (11 patients), mental changes (12 patients) and orthostatic hypotension (8 patients). Although lergotrile, when added to levodopa, has a definite antiparkinsonian effect, the incidence of adverse effects, particularly hepatotoxicity, makes it unlikely that this ergot alkaloid will become widely available for the treatment of PD. Analogues of lergotrile have been synthesized, and it is hoped that they will duplicate the antiparkinsonian effect of this drug without its toxicity.

[Conditions of exolipase biosynthesis by the fungus Oospora fragrans].

The fungal strain Oospora fragrans isolated from sunflower seeds had a high lipase activity, especially on the media containing soybean flour and various oils. The fungal lipase showed inductive characteristics, the best inducers being olive and soybean oils. The biosynthesis of lipase from O. fragrans occurred at the optimal temperature of 26-30 degrees C and strong aeration. Maximun of lipolytic activity did not coincide with maximum accumulation of biomass and developed at the log-phase of the fungal growth. With age the lipid content in the mycelium increased in parallel with the biomass accumulation. Mycelial lipids of O. fragrans contained nine fractions, among which fatty acids and triglycerides were in predominance. The study of properties of O. fragrans lipase showed a high pH value and thermal stability, the capacity to function at low temperatures, and activity towards many substrates. The enzyme readily hydrolyzed not only natural vegetable (olive, linseed and mustard) oils but also oils of microorganisms grown on petroleum hydrocarbons.

Distribution and density of mosquitoes in two endemic areas for bancroftian filariasis in Sorsogon, Philippines.

Mosquito density in Rangas where abaca is in abundance is much higher, almost twice, than that of Putiao where abaca is absent. The adult density of Aedes poecilus over Aedes ananae in the two areas combined is 3 to 4 times whereas the larval density of the former is much lower than Aedes ananae. The banana axils is a favorite breeding place for Aedes poecilus but may also utilize the abaca axils. This finding is very favorable in the transmission of bancroftian filariasis because this species of banana is planted around houses even closer to human dwelling than the abaca plants. Aedes poecilus being more anthropophylic than zoophylic can be domesticated as Culex quinquefasciatus and Aedes aegypti which again is a factor in favor of ideal transmission of the disease. The fact that bananas are planted around houses both in villages and towns, transmission of the disease could easily occur in both places, which should be borne in mind when planning a control program. The number of dissected mosquitoes is too small to be of significance in vector determination.

[Acid-base equilibrium in mother and fetus during labor and the resistance of newborn calves].

Investigated were a total of 59 cows presenting normal processes, of giving birth as well as dystocia, the time of delivery lasting 2, 10, 24, and 36 hours. Seventeen cows with dystocia were treated intravenously with 500 cm3 of a 4 per cent solution of sodium bicarbonate and 40 g glucose. It was found that the changes in the pH values, alkali reserves, glycogen, and the peroxydase activity of the neutrophiles in the mothers and fetuses were dependent on the duration of parturition. Cases of difficult labour with a prolonged calving period led to lowering the pH values the alkali reserves, and the enzyme activity of leukocytes both in the cows and the newborn calves. Newborns delivered 24 and 36 hours from the beginning of the parturition act showed low resistance and had weakly expressed phagocytic and bactericidal blood activity. The intravenous application of 50 ml of a 4% solution of sodium bicarbonate and 40 g glucose to cows with dystocia up to the 10th day led to a compensatory response on behalf of the acid-base balance in the calves.

[Comparative analysis of the morphogenesis of the Getah virus in cell cultures of vertebrates and mosquitoes].

Reproduction of Getah virus (the genus Alphavirus) in piglet kidney (PK) and A. aegypti mosquito cell cultures was studied morphologically. Inoculation of PK cells resulted in the development of a lytic infection. Virus morphogenesis in these cells showed the features known for other alphaviruses. In mosquito cell cultures persistent infection developed which was not accompanied by any marked changes in morphology or proliferative activity of the culture. All the morphological and cytochemical data indicate that a certain role in the establishment and maintenance of virus persistence in A. aegypti cell culture may be played by exo- and endophagocytic processes owing to which a peculiar "self-purification" of the cells from the virus and its components occurs. Incubation of the persistently infected culture of mosquito cells at a suboptimal temperature resulted in a marked reduction in the number of newly formed virions which sharply increased with the shift up to the optimal temperature. The experimental results give some insights into certain aspects of alphavirus ecology.

Cellulose-decomposing fungi.

The present article gives a survey of the cellulose-decomposing fungi. It is concerned with the micro-organisms having the capability of degradating cellulose sources. It includes the factors influencing cellulose-decomposing fungi, cellulose-decomposing enzymes, mechanisms of degradation, and factors influencing the cellulolytic enzymes (cellulases).

Gastro-oesophageal reflux after surgical treatment of hiatal hernia with and without severe reflux complications. A follow-up study.

One hundred and sixteen patients operated upon for hiatal hernia with gastro-oesophageal reflux and with or without reflux complications were postoperatively examined by personal interview, X-ray study, pH measurements and study of the oesophageal motility 1 to 10 years postoperatively. The patients without severe reflux complications were operated upon mainly with a modified Husfeldt hernia repair and the patients with complications, such as oesophageal stricture and shortening, underwent various surgical procedures. The main reason for unsatisfactory clinical results, with persistent reflux symptoms, was gastro-oesophageal reflux uncorrected by the surgical procedure. However, gastro-oesophageal reflux was detected even in completely asymptomatic patients. It was found that the reflux symptoms were influenced by the oesophageal motility. The clinical results were better and recurrence of hernia and the occurrence of pathological reflux were lower in patients operated upon for hernia without severe reflux complications. Creation of a competent antireflux barrier between the oesophagus and stomach for control of gastro-oesophageal reflux is much more difficult in patients with severe reflux complications.

Delirium tremens and related clinical states. Aetiology, pathophysiology and treatment.

Definitions of Delirium Tremens (DT) and related clinical states are discussed together with the concepts of aetiology and pathogenesis in relation to psychiatric disease. The withdrawal theory which considers reduction or cessation of alcohol intake as an important precipitating factor in DT is discussed; this theory is supported by experimental studies of ethanol withdrawal in man and by studies indicating cross dependence between ethanol and several other CNS depressors; the arguments in the literature for and against the withdrawal theory are discussed. Other possible aetiological factors such as type of liquor, hypovitaminosis, liver disease, dysfunction of the adrenals and fat emboli are reviewed, and it is concluded that these factors are unimportant as precipitating or specific aetiological factors in DT. Possible pathophysiological mechanisms are discussed, especially concerning electrolyte metabolism and cerebral blood flow and oxygen consumption. It is concluded that the pathophysiology of the disease is poorly understood, and some possibilities for future research are indicated. The discussion about treatment of DT is concentrated on drug treatment, and the literature concerning antipsychotics and sedatives is reviewed. It is concluded that barbital, a long-acting barbiturate, is the most effective treatment; diazepam can be recommended as an acceptable alternative. Finally, practical recommendations concerning treatment are given.

A scoring system for nonstressed antepartum fetal heart rate monitoring.

A scoring system developed to assess nonstressed antepartum fetal heart rate records has been evaluated. The system scores from 0 to 2 for each of five factors, i.e., baseline rate, amplitude and frequency of fluctuation, decelerations, and accelerations. A total of 1,367 nonstress tests in 284 patients were retrospectively reviewed. Three groups of patients were delineated with respect to the fetal heart rate scores. When the group with the highest score of 8 to 10 and a perinatal mortality rate (PNM) rate of 4.7/1,000 was compared with the groups having intermediate (5 to 7) and low (less than or equal to 4) scores, there was a highly significant increase in PNM to 69.8/1,000 (p less than 0.01) and 433/1,000 (p less than 0.001), respectively. A significant increase in low Apgar scores at 1 and 5 minutes, in fetal acidosis (low umbilical arterial pH), and in duration of intensive care nursery stay was also seen as the fetal heart rate score progressively deteriorated from the 8 to 10 group to the less than or equal to 4 group. We conclude that this scoring system clearly delineates normal from prepathologic and pathologic changes in fetal heart rate and can be used as warning of impending fetal death.

Pressor response buffering by beta-adrenergic and cholinergic vasodilation in tranquilized dogs.

Buffering of alpha-receptor-mediated pressor responses by beta-adrenergic or cholinergic vasodilation in tranquilized, chronically instrumented gos was investigated. Increases in aortic pressure were produced in the same animal by intravenous injections of phenylephrine in the control state and in three successive experimental states by 1) pacing the heart to remove the reflex capability to lower heart rate, 2) pacing the heart and beta-blockade to remove beta-adrenergic vascular buffering, and 3) beta-blockade plus atropine to also remove cholinergic vascular buffering. The pressor response in each experimental state was greater than that in the state preceding it. With the combined beta-adrenergic and cholinergic blockade, the pressor response to an alpha-receptor stimulation was three times greater than that of the control state. From an analysis of the components of the pressor response, cardiac output, and peripheral resistance, it is suggested that normal buffering of an alpha-mediated pressor response may include beta-adrenergic and cholinergic vascular dilation in addition to a decrease in heart rate.

Converting-enzyme activity and pressor responses to angiotensin I and II in the rat awake and during anesthesia.

Plasma renin activity (rate of angiotensin I generation) does not increase during anesthesia with ketamine, fluroxene, halothane or enflurane in the sodium-repleted rat. However, blood pressure decreases when an angiotensin II antagonist, saralasin, is administered during halothane or enflurane anesthesia, but not during ketamine or fluroxene anesthesia. Differences in the rates of conversion of angiotensin I to angiotensin II induced by various anesthetic agents could help explain these previous findings. To determine the effects of anesthetic agents on angiotensin I conversion, experiments were performed in vitro and in vivo. The activities of rabbit pulmonary converting enzyme in the presence and absence of halothane or fluroxene were measured as rates of appearance of the dipeptide, histidyl-leucine, a product of angiotensin I hydrolysis to angiotensin II. Halothane and fluroxene did not alter conversion. Infusions of angiotensin I and angiotensin II were given to Wistar rats to construct dose-blood pressure response curves. The animals were then anesthetized with ketamine or halothane and infusions were repeated. Angiotensin I and angiotensin II induced similar blood pressure responses in awake and anesthetized rats. However, ketamine accentuated the pressor responses to angiotensin I and angiotensin II, whereas halothane depressed the responses. With the anesthetic agents studied, there is no significant effect on conversion of angiotensin I to angiotensin II either in vitro or in vivo.

[The effects of dobutamine in postoperative disorders of left ventricular function in coronary patients undergoing abdominal surgery. Apropos of 18 cases].

Increasing doses of dobutamine were administered to the first group of 10 coronarians having undergone and abdominal surgical procedure, and presenting, one hour after awakening from the anesthesia, hemodynamic modifications with a diminution of cardiac index (CI), systolic index (SI), systolic work index of the left ventricle (SWILV) increase in the pulmonary capillary pressure (PCP), and in the total peripheral resistance (TPR), as well as an acceleration of the cardiac rate (CR). Doses of dobutamine of 5 or 7.5 microgram.kg-1.min-1 corrected the IC, PCP and TPR. Dobutamine ameliorated the SI and SWILV in an increasing fashion up to a dose of 10 microgram.kg-1.min-1 only and without restoring them to the control values of the pre-operative period. CR progressively increased with the increasing of the doses reaching 126 +/- 21.5 beats min-1 for 15 microgram.kg-1.min-1. Extrasystoles appeared at dose levels of 12.5 and 15 microgram.kg-1.min-1 in two patients. Tests of vascular filling (pre-charge tests) carried out in the second group of patients under 10 microgram,kg-1.min-1 of dobutamine and in a third group under 15 microgram.kg-1.min-1 showed a good cardiac adaptation to filling, equal or superior to that of the pre-operative period. It also appeared that the amelioration of CF obtained with a moderate vascular filling (300 ml of low molecular weight dextran) under 10 microgram.kg-1.min-1 of dobutamine is greatly superior to the amelioration obtained by 10 to 15 microgram.kg-1.min-1 of dobutamine.

[Focalized irrigation-lavage and sequential use of a new antiseptic by local and general route. Preliminary report apropos of 2 cases of suppurative pancreatic necrosis].

In view of the severe course seen in the presence of any suppurated pancreatic necrosis, it was felt to be of value to treat two patients by the adjuvant use of a new antiseptic tauroline, administered locally and, where appropriate, systemically. After surgical excision of pancreatic and peripancreatic necrotic tissue, the authors prefer to avoid active suction drainage of the residual pocket, replacing it by continuous and prolonged post-operative irrigation-lavage, together with the sequential, twice-daily use of a solution of 2 p. 100 tauroline. In view of the presence of a Gram negative septicemia (organisms identical to those of the suppurated area), the same substance was given by intravenous infusion in a 0.5 p. 100 solution, this being the only systemic, anti-infectious therapy. The effectiveness, good tolerance and originality of this new substance, active against Gram negative, Gram positive, aerobic and anaerobic organisms as well as yeast, have led the authors to immediately report their initial experience with its use.

Relationship between arterial and venous bicarbonate values.

We determined the clinical efficacy of using the venous CO2 value, as obtained with routine "electrolytes", in acid-base management. Venipuncture samples for venous CO2 content and chloride concentrations were obtained in 336 patients with arterial blood pH, PaO2, PaCO2, and oxygen saturation determinations. The linear correlation between actual calculated arterial HCO3- and the measured venous CO2 was significant (P less than .001). Using venous CO2, chloride, and arterial pH values, we present various prediction equations for estimating arterial HCO3-. We determined the effects of time delay, exposure to air, and acute changes in arterial blood pH and PaCO2 on venous CO2 levels. Venous CO2 determinations should not be substituted for the arterial HCO3 value in the Henderson-Hasselbalch equation to calculate arterial blood pH or PaCO2. Clinically, the venous CO2 value has little direct use, but when venous CO2 content is abnormal, it should alert the clinician to the need for obtaining arterial blood gas and pH values.

Antigenic and immunogenic properties of gamma-glutamyl-transferase preparations.

Incubation time of human kidney gamma-glutamyl-transferase (GGT) with specific antiserum and precipitate formation caused no effect on inhibition of the enzyme activity. In the obtained precipitate GGT activity was independent of the amount of antiserum and contained 30% of the initial enzyme activity. After reaction of the serum with human liver GGT or with the urine enzyme a precipitate formation and decrease in the enzyme activity was noted. In the serum incubated with GGT isolated from animal kidneys, neither inhibition nor precipitation was observed. The precipitate of bovine kidney GGT with a specific antibody independently of the amount of antibody contained 50% of activity. After treatment of bovine GGT with bromelain four protein fractions giving precipitin lines in crossed-immunoelectrophoresis were observed but only one of them preserved catalytic activity. Immunogenicity of newly obtained immobilized GGT preparations was much lower than that of the native enzyme. A distinct and different influence of antibody on the affinity of native and immobilized GGT preparations to donor and acceptors of gamma-glutamyl group was noted. The immunofluorescent technique was used for GGT localization in some bovine tissue sections.

The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose.

Equilibrium and kinetic aspects of the binding of several proteins to N-(3-carboxypropionyl)aminodecyl-Sepharose, an amphiphilic ampholytic adsorbent, were studied at 22 degrees C, pH 7.0, I 0.10--0.12. In the absence of detergents Scatchard plots are linear for human haemoglobin and soya-bean trypsin inhibitor, but non-linear for bovine serum albumin, which is also adsorbed more tightly than the other two proteins. The introduction [corrected] of 3.5mM-sodium dodecyl sulphate causes dramatic increases in the amounts and affinities of serum albumin and haemoglobin adsorbed, but has relatively little effect on the trypsin inhibitor. At concentrations of sodium dodecyl sulphate greater than about 10mM there is a fall in the binding of all proteins, owing to competition from the detergent for binding sites on the adsorbent, and a tendency towards more uniform behaviour by different proteins. Kinetic experiments suggest that in the absence of the detergent haemoglobin and serum albumin are adsorbed initially by mainly ionic forces, but that subsequently hydrophobic forces become dominant. Addition of 3.5 mM-sodium dodecyl sulphate causes pronounced changes in the time course of adsorption of haemoglobin and serum albumin, the nature of the changes being different for each protein. The significance of these results is discussed.

Kinetics and mechanism of action of aldehyde reductase from pig kidney.

An improved procedure for purifying aldehyde reductase is described. Utilization of Blue Dextran--Sepharose 4B and elimination of hydroxyapatite chromatography greatly improves the yield and ease of purification. Starting with 340 g of kidney tissue (two pig kidneys) approx. 50 mg of purified reductase may be routinely and reproducibly obtained. The purified reductase was used to establish the kinetic reaction mechanism of the enzyme. Initial-velocity analysis and product-inhibition data revealed that pig kidney aldehyde reductase follows an Ordered Bi Bi reaction mechanism in which NADPH binds first before D-glyceraldehyde. The limiting Michaelis constants for D-glyceraldehyde and NADPH were 4.8 +/- 0.7 mM and 9.1 +/- 2.1 micrometer respectively. The mechanism is similar to that of another monomeric oxidoreductase, octopine dehydrogenase, towards which aldehyde reductase exhibits several similarities, but differs from that of other aldehyde reductases. Phenobarbital is a potent inhibitor of aldehyde reductase, inhibiting both substrate and cofactor non-competitively (Ki = 80.4 +/- 10.5 micrometer and 66.9 +/- 1.6 micrometer respectively). Barbiturate inhibition seems to be a common property of NADPH-dependent aldehyde reductases.

Evaluation of the effects of clobazam, A 1,5 benzodiazepine, on mood and psychomotor performance in clinically anxious patients in general practice.

1 The methodology of clinical trails of anxiolytic drugs carried out in general practice conditions is discussed. Particular problems include: the selection of suitable patients; placebo effects; the influence of non-specific variables such as life-events; and patient compliance. 2 Clobazam, a novel 1,5 benzodiazepine, and diazepam were compared with placebo in a double-blind group comparative trial in general practice, which was designed to avoid as many confounding variables as possible. Anxiety-reducing effects were evaluated and at the same time the effects of the drugs on psychomotor performance were examined. 3 Both clobazam and diazepam produced significant improvements in anxiety ratings on the Hamilton Anxiety Scale and the Morbid Anxiety Inventory, whereas placebo did not. 4 The placebo group demonstrated a significant improvement in performance on a pursuit rotor and digit symbol substitution test (DSST), whereas the diazepam group's performance did not change. The clobazam group showed improvement in both tests, significantly so in the DSST. This suggests that diazepam produces impairment in performance, which had negated the practice effects seen in the placebo group, whereas clobazam did not seem to produce similar impairment.

Some aspects of the effects of clobazam on human psychomotor performance.

1 Three studies are described, the first being a comparison of the effects of acute night-time doses of clobazam 20 mg, amylobarbitone sodium 100 mg, nitrazepam 5 mg and placebo, on choice reaction time, critical flicker fusion (CFF) and stabilometer performance. Clobazam improved early morning performance on a choice reaction test, in contrast to the other two active drugs. 2 Repeated doses of clobazam 10 mg three times daily, chlordiazepoxide 10 mg three times daily and diazepam 5 mg three times daily were given for 5 days. Again clobazam did not produce any impairment of psychomotor performance, and noticeably increased CFF thresholds. 3 The effects of an acute night-time dose of clobazam 20 mg on psychomotor performance the morning after night-time medication were correlated with the neuroticism scores (on the EPI) of the subjects. Clobazam exerts a differential effect on psychomotor performance dependent on the basic personality trait. 4 Clobazam seems to differ significantly from the 1,4-benzodiazepines in that, although it reduces anxiety, it does so without any apparent impairment of psychomotor performance.

Conformational transitions and vibronic couplings in acid ferricytochrome c: a resonance Raman study.

Resonance Raman spectral changes in ferricytochrome c as a function of pH between 6.7 and 1.0 are reported and the structural implication is discussed in terms of the "core-expansion" model advanced by L. D. Spaulding et al. [(1975) J. Am. Chem. Soc. 97, 2517]. The data are interpreted as indicating the iron in high-spin ferricytochrome c (at pH 2.0) with two water molecules as axial ligands lies in the plane of the porphyrin ring. At pH 1.0 there is a different high-spin form of cytochrome c which has an estimated iron out-of-plane distance of approximately 0.46 A. The effect of a monovalent anion at pH 2.0 is to produce a thermal spin mixture with predominant low-spin species. Excitation at approximately 620 nm in acid cytochrome c (pH 2.0) enhances only three depolarized ring vibrations at 1623, 1555, and 764 cm-1. Marked enhancement of depolarized modes relative to polarized and anomalously polarized modes is attributed to the vibronic coupling between porphyrin pi leads to pi and porphyrin pi leads to iron (dpi) charge-transfer states.

[Peroxidase activity of hemoglobin, modified at the carboxylic groups of heme and amino acids].

The effects of modification of heme carboxylic groups by omega-aminoenantic acid and L-phenylalamine on the peroxidase activity of hemoglobin were studied. For this purpose the peroxidase activities of the original compounds--hemin, hemin-aminoenantic acid, hemin-phenylalanine and hemoglobins prepared from the hemin and globin compounds--hemoglobin, aminoenantyl-hemoglobin and phenylalanine hemoglobin--were determined. The dependence of the peroxidase activity of these compounds on their concentrations and pH was analyzed. It was shown that 40--50% modification of the heme carboxylic groups by amino acids decreases the peroxidase activity of the modified hemins and that of modified hemoglobins reconstructed from these hemins and globin. A decrease of the catalytic activity of the hemoglobin derivatives is due to a lower peroxidase activity (as compared to hemin) of the modified hemins. It is thus concluded that the amino acid modification of the carboxylic groups of heme does not affect the heme-protein interactions in the hemoglobin molecule.

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.

A comparison of reaction conditions for the automated determination of gamma-glutamyltransferase activity in serum.

The methods of Rosalki and Tarlow and the Scandinavian Committee on Enzymes for the determination of gamma-glutamyltransferase activity in serum have been compared, with an automatic reaction-rate analyzer in which the reaction is initiated by the addition of a concentrated solution of gamma-glutamyl-p-nitroanilide. Results are approximately 4% lower by the Scandinavian method, because of its lower substrate concentration. However, both methods are of comparable reproducibility. The greater stability of the more dilute substrate solution specified in the Scandinavian method has been confirmed, but attempts to stabilize solutions of gamma-glutamyl-p-nitroanilide at the higher concentrations recommended by Rosalki and Tarlow by the addition of organic solvents were accompanied by some enzyme inhibition. The comparative instability of the substrate solutionof Rosalki and Tarlow is unlikely to lead to erroneous results; however, the Scandinavian formulation offers the advantage of a more economical use of reagents, at the expense of a small and probably unimportant reduction in sensitivity.

Effects of changes in arterial carbon dioxide tension on oxygen consumption during cardiopulmonary bypass.

The effects of changes in arterial carbon dioxide tension (PaCO2) on the oxygenation of tissues in 34 patients undergoing surgery for aortocoronary bypass were studied while temperature, systemic blood flow, and the delivery of oxygen to the peripheral tissues remained constant. Mixed venous and superior vena caval oxygen tensions (PvO2 and PsvcO2, respectively) and oxyhemoglobin saturations and the in vivo partial pressure of oxygen at which 50 percent of the hemoglobin is saturated (P50) increased with PaCO2, while peripheral vascular resistance, in vitro P50, the level of 2,3-diphosphoglyceric acid in the red blood cells, and the level of lactate in the blood remained constant. There was a close correlation between increases in PaCO2 and increases in PvO2 (r = 0.912; P less than 0.001) but not increases in PsvcO2 (r = 0.364; not significant). This indicated that the total-body consumption of oxygen diminished with increases in PaCO2 but that some regional redistribution of oxygen consumption occurred between the superior and inferior vena caval vascular beds. Since the level of lactate in the blood remained constant and since signs of metabolism acidosis did not develop, the reduced oxygen consumption due to increases in PaCO2 did not result in detectable increases in anaerobic metabolism.

Diastereoisomeric glucuronides of oxazepam. Isolation and stereoselective enzymic hydrolysis.

Oxazepam glucuronide isolated from swine urine by previously published methods was separated into its diastereoisomers by ion-exchange chromatography on a preparative scale. Quantitative high-performance liquid chromatography was used to monitor the separation. The two isomers were obtained in analytically pure form and then characterized by elemental analysis, oxazepam content, mass spectrometry, ultraviolet spectroscopy, optical rotation and optical rotatory dispersion-circular dichroism. The latter permitted the assignment of the dextrorotatory and the levorotatory isomers to the (S)- and (R)- configurations, respectively. Rates of enzymic hydrolysis depend on the configuration of the substrate as well as on the enzyme preparation used. Rate of cleavage was highest with the (S)-(+)-glucuronide and beta-glucuronidase from Escherichia coli. This enzyme possesses the highest degree of stereoselectivity; it hydrolyzes the (S)-(+)-isomer more than 400 times faster than the (R)-(-)-form. Bovine liver glucuronidase is less stereoselective, whereas glucuronidase preparations of molluscan origin exhibit little stereoselectivity. The ready hydrolysis of one of the glucuronides by an enzyme from an intestinal microorganism may play a role in the enterohepatic circulation of oxazepam.

Immunoreactive somatostatin in the hypothalamus and other regions of the rat brain: effects of insulin, glucose, alpha- or beta-blocker and L-dopa.

Effects of various hormonal and pharmacological manipulations on somatostatin distribution were investigated to elucidate the physiological significance of somatostatin in the hypothalamus and the other regions of the rat brain. Immunoreactive somatostatin (IRS) was measured by radioimmunoassay newly developed. Insulin induced an increase of hypothalamic IRS and a decrease of plasma RGH, while glucose administration resulted in the opposite responses, which were not significant. Insulin also increased IRS in the thalamus and the brain stem. The insulin-induced increase of hypothalamic IRS was reduced by hyperglycemia. Glucagon reduced IRS initially and then increased it with an elevation plasma RGH. L-dopa did not affect hypothalamic IRS, although it decreased plasma RPRL. Phentolamine slightly increased plasma RGH and decreased IRS in most regions of the rat brain, while propranolol increased IRS in these regions. Pretreatment with propranolol significantly increased plasma RGH 120 min after insulin administration, and hypothalamic IRS decreased initially by pretreatment with propranolol, and then it increased significantly. When pretreated with propranolol, glucagon markedly increased plasma RGH and decreased IRS significantly. From these findings it is concluded that hypothalamic IRS may participate in the hormonal regulatory system in correlation to plasma RGH, as observed in studies on plasma GH and hypothalamic IRS following insulin, glucose, propranolol or phentolamine administration, but IRS in other regions of the brain may have some other actions as a neurotransmitter or a modulator, because of no significant correlation between plasma GH or PRL and IRS in these regions following various stimuli. In addition, glucose homeostasis and adrenergic mechanism may be important factors in regulating IRS in the rat brain.

Kinetic analysis of the interaction of the phosphatidylcholine exchange protein with unilamellar vesicels and multilamellar liposomes.

The mode of action of the phosphatidylcholine exchange protein from bovine liver has been studied by using unilamellar vesicles and multilamellar liposomes both of which membranes contain phosphatidylcholine and phosphatidic acid. The protein-mediated exchange of phosphatidylcholine between vesicles and liposomes fit the kinetic model presented in a previous study [V.D. Besselaar et al. (1975) Biochemistry, 1j, 1852]. Kinetic analysis of the rates of exchange indicate that the apparent dissociation constant of the exchange protein-vesicle complex decreases with an increasing phosphatidic acid content of the vesicles. Both vesicles and liposomes of 10 mol% phosphatidic acid show the same dissociation constant; on the other hand, both the formation and the disruption of the protein-membrane complex was 50--100-times higher for the vesicles than for the liposomes. This implies that the exchange protein can discriminate between vesicles and liposomes. Equilibrium gel chromatography of a column of Bio Gel A-5m confirmed that the exchange protein binds more strongly to vesicles of an increased phosphatidic acid content. The protein-mediated exchange of phosphatidylcholine in the vesicle-liposome system demonstrates a pH optimum at 4.0 to 5.5. The kinetic analysis at pH 5.0 as compared to pH 7.4 indicates that the enhanced exchange at pH 5.0 can solely be accounted for by altered interaction of the exchange protein with the liposomes.

The value of simple tests in the detection of human ovulation.

Cervical mucus viscosity (CMV) and fern test (FT), leukocyte alkaline phosphatase (LAP) activity, basal body temperature (BBT) and serum luteinizing hormone (LH) levels were determined during 31 normal menstrual cycles of 29 volunteers. The day of the serum LH surge was taken as a reference point in evaluating the reliability and sensitivity in predicting ovulation of the other tests studied. Serum LH surge was accompanied by a sudden decrease in CMV, an increase in LAP activity, a gradual increase in FT and biphasic changes of BBT. In about 95% of cycles studied, the lowest values of CMV and peak values of LAP activity appeared on the day of the serum LH surge or one day before or one day after. The lowest point of the BBT coincided with the LH surge in only 26% of cycles. In 53% of cycles, the span was greater than one day before or after the LH peak. The FT corresponded to the LH surge in only 33% of the cycles, while in an additional 33% the preovulatory FT response occurred more than one day before or after the LH surge. The results indicate that CMV and LAP tests are rapid, simple and reliable for monitoring follicular maturation and the timing of ovulation when carried out daily, and may be of value in monitoring treatment during the induction of ovulation.

Fibrinolytic activity of menstrual blood in normal and menorrhagic women and in women wearing the Lippes Loop and the Cu-T (200).

Assessment was made of the fibrinolytic activity in menstrual and peripheral blood of 30 normally menstruating and 30 menorrhagic patients and of 30 women wearing Lippes Loops and 15 wearing CU-T (200)s. Assessment was performed by measuring the area of lysis on heated and unheated fibrin plates. Also, histochemical identification of fibrin fibrils in the menstrual endometrium was performed by Mallory's phosphotungstic acid hematoxylin method. Results in the normally menstruating group were compared to those of the menorrhagic women, and together these were compared with results in the groups of women wearing intrauterine devices. The fibrinolytic activity in menstrual blood was significantly increased in menorrhagic patients compared to that in normally menstruating patients, but no significant difference was detected in the plasma of either group. The histochemical study of the normally menstruating endometrium revealed dense intravascular and extravascular deposits of fibrin. Less dense intravascular fibrin deposits, but no extravascular ones, were present in the menorrhagic patients. The increase in the fibrinolytic activity of menstrual blood and the decrease in the density of fibrin deposits in the menstrual endometrium of the menorrhagic women were thus associated and were probably involved in the excessive menstrual loss. The fibrinolytic activity of menstrual blood in women wearing Lippes Loops was higher than that in women with Cu-T (200)s and was associated with decreased density of fibrin fibrils in the menstruating endometrium. This may explain the increased blood loss associated with the use of the Lippes Loop.

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.

The effects of X-irradiation on lens reducing systems.

Studies have been made of the effects of X-ray on various lens reducing systems, including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS) and of certain enzymes, including GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PG). It was found that during several weeks following X-irradiation but prior to cataract formation, there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the X-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the X-rayed lens was found to decrease significantly relative to controls 1 week prior to cataract formation, and the ratio of NADPH to NADP+ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the X-rayed lens. A corresponding decrease occurred in the activity of the HMS in X-rayed lenses as measured by culture in the presence of 1-14C-labeled glucose, G-6-PD was partially inactivated in the X-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to X-irradiation. The data indicate that X-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high-molecular-weight protein aggregates may be initiated.

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.

Uptake of circular deoxyribonucleic acid and mechanism of deoxyribonucleic acid transport in genetic transformation of Streptococcus pneumoniae.

Deoxyribonucleic acid (DNA) from the covalently closed circular DNA molecules of Pseudomonas phage PM2 was found to enter normally transformable cells of Streptococcus pneumoniae as readily as linear bacterial DNA. In a mutant of S. pneumoniae that lacks a membrane nuclease and is defective in DNA entry, as many molecules of PM2 DNA as of linear DNA were bound on the outside of cells at equivalent DNA concentrations. Bound DNA suffered single-strand breaks, but circular DNA with preexisting breaks was bound no better than closed circles. In the presence of divalent cations, DNA bound to cells of a leaky nuclease mutant showed double-strand breaks. At least the majority of PM2 DNA that entered normal cells was single stranded. These results are consistent with a mechanism for DNA entry in which DNA is first nicked on binding, then a double-strand break is formed by cleavage of the complementary strand, and continued processive action of the membrane nuclease facilitates entry of the originally nicked strand. Although the bulk of circular donor DNA appeared to enter in this way, the results do not exclude entry of a small amount of donor DNA in an intact form.

The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer.

A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.

Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase.

The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.

Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure.

We have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.

Interaction of amiloride and lithium on distal urinary acidification.

The interaction of amiloride and LiCl administration on renal HCO3 handling was studied in hydropenic rats. Amiloride administration resulted in a significant increase in Na, Cl, and HCO3 excretion, whereas K excretion decreased significantly. LiCl administration resulted in a significant increase in Na, Cl, K, and HCO3 excretion. LiCl administration to animals receiving amiloride led to a significant increase in HCO3 excretion but failed to cause an increase in Na or K excretion. Addition of amiloride to animals receiving LiCl resulted in a significant increase in Na and HCO3 excretion. The net increase in fractional HCO3 excretion seen in this group was greater than that seen in all other groups. The finding that the net increase in FEHCO3 was greater in animals receiving amiloride after administration of LiCl than in animals receiving LiCl after amiloride administration indicates that amiloride blunted the effect of LiCl on HCO3 excretion. Administration of amiloride to both normal rats and to rats infused with Li during HCO3 administration resulted in a significant decrease in U-B Pco2 which could not be explained by the decrease in urine HCO3 concentration. These data demonstrate that amiloride inhibits distal acidification in vivo. LiCl administration also resulted in a decrease in U-B Pco2 which could be explained by the decrease in urine HCO3 concentration. LiCl administration also resulted in a decrease in TcH2O which could be prevented by prior administration of amiloride. These data indicate that amiloride blunts the effect of LiCl on urinary acidification, an effect similar to that observed on urinary concentration. These data suggest that the effect of Li on urinary acidification is in part dependent on Li entry into the cell.

Acute and chronic methadone exposure in adult rats: studies on arterial blood gas concentrations and pH.

The effects of different dosages of methadone on respiration were determined by evaluating arterial blood pCO2, pO2 and pH in naive and opioid-addicted animals. Male Sprague-Dawley rats were treated (i.p.), acutely or chronically, with either 2.5, 5.0 or 7.5 mg/kg of dl-methadone hydrochloride; appropriate saline controls were utilized. Blood was sampled from the tail artery before injection and 15, 30, 60, 120, 180 and 240 min postinjection. Animals exposed to methadone in acute experiments exhibited a respiratory depression that involved hypoxemia, hypercapnia and/or acidosis. In addition, the magnitude of this respiratory depressant action was dose-dependent and reached a maximal point 15 to 30 min after drug administration. Rats receiving chronic methadone exposure showed few alterations from control blood gas concentrations and pH. This study demonstrates that acute methadone administration is associated with respiratory depression, with the extent of reductions in pCO2, pO2 and pH related to drug dosage. In addition, chronic methadone treatment confers a substantial tolerance to the respiratory depressant action of methadone.

Effects of potassium depletion on renal tubular chloride transport in the rat.

Potassium depletion (KD) causes renal chloride-wasting. To investigate the effects of KD on renal tubular reabsorption of chloride, balance, clearance, micropuncture, and microinjection studies were performed on potassium-depleted rats. KD was produced by omitting potassium from the diet and by administration of DOCA on days 2 and 3; rats were studied on days 9 to 12. Diets were chloride-free in both control and KD groups. In the KD group, balance experiments confirmed greater chloride depletion and continued chloride-wasting, and clearance studies showed an increased FECl. Muscle potassium was reduced by 27% as compared to control. Whole kidney and single nephron GFR were reduced in KD rats to 72 and 74% of control. Fractional (6 +/- 6% vs. 22 +/- 4%, P less than 0.05) and absolute chloride reabsorption in the proximal tubule was not different. Fractional reabsorption of delivered chloride was reduced in the loop of Henle (92 +/- 0.8% in KD vs. 95 +/- 0.7% in control, P less than 0.02). Transtubular chloride ratio (0.28 +/- 0.02 vs. 0.21 +/- 0.02, P less than 0.02) was increased at the early distal tubule. Fractional delivery of chloride (8 +/- 0.9 vs. 5 +/- 0.5%, P less than 0.02), and fluid (26 +/- 1 vs. 22 +/- 1%, P less than 0.05) were also increased in KD at the early distal tubule. Recovery of chloride 36 injected into late distal tubules was 88 +/- 1% on a normal chloride intake, 62 +/- 2% in chloride depletion, and 88 +/- 2% in potassium and chloride depletion. Thus, KD depresses chloride reabsorption in the proximal tubule and in the loop of Henle, and it decreases chloride 36 efflux from the collecting duct.