Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies.


Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum. Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment.


Mayo Seminars in Psychiatry: dopamine and schizophrenia--a review. Considerable evidence exists linking dopamine with schizophrenia. Other neurotransmitters including acetylcholine, serotonin and gamma-aminobutyric acid (GABA) are also being implicated. Neurotransmitters act via receptors on brain cells and evidence suggests that antipsychotic drugs exert their therapeutic actions and produce side effects by receptor blockade.


Kinetics of hydrolysis of phenylthiazolones of arginine, homoarginine, norarginine, and canaavanine by trypsin. Phenylthiazolones (PTAs) of arginine and its homologs and analogs, homoarginine, norarginine (alpha-amino-gamma-guanidinobutyric acid), canavanine, and gamma-hydroxyarginine, were prepared. A steady-state kinetic analysis of the trypsin [EC 3.4.21.4]-catalyzed hydrolysis reactions was carried out and the kinetic parameters for these internal thioesters were compared with those for normal linear ester substrates. PTA-gamma-hydroxyarginine was so labile that hydrolysis by the enzyme could not be followed. PTA-arginine has a specificity constant (Kcat/Km) comparable to that for the Nalpha-unblocked arginine ester substrate, though the value is about 0.1% of that for a specific ester substrate, Nalpha-tosylarginine methyl ester. PTA derivatives of canavanine and homoarginine were hydrolyzed with Kcat/Km walues of the same order of magnitude as that for PTA-arginine. However, PTA-noraginine was much less susceptible to tryptic hydrolysis that PTA-homoarginine, while the linear esters of norarginine are known to be more susceptible than those of homoarginine.


Differences between the reactivities of two pyridine nucleotides in the rapid reduction process and the reoxidation process of adrenodoxin reductase. The reaction process of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (Kobs values were 20.5 and 16.0s-1). These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermidiate is caused by a two-electron transfer process from the reduced pyridine nucleotide to the enzyme flavin. On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semiquinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR measurement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complex with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.


Extrusion of sodium ions energized by respiration and glycolysis in Escherichia coli. Extrusion of sodium ion from cells of Escherichia coli was measured using an Na+ electrode. When oxygen was supplied to an anaerobic cell suspension, extrusion of Na+ was observed. The addition of glucose under anaerobic conditions also caused Na+ efflux. The extrusion of Na+ energized by respiration and glycolysis was completely inhibited by a proton conductor, carbonyl cyanide m-chlorophenylhydrazone. These observations are consistent with the view that Na+ transport occurs secondarily to H+ circulation. Interestgly, induction of the melibiose transport system, which is coupled to Na+, greatly enhanced Na+ transport activity.


Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts. The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.


Magnesium-sensitive guanylate cyclase and its endogenous activating factor in Tetrahymena pyriformis. Guanylate cyclase [EC 4.6.1.2] activity in Tetrahymena pyriformis cells was associated with particulate fractions, but not with soluble fractions. Mg2+ was much more effective than Mn2+ in activating the cyclase activity. Both specific and total cyclase activities with Mg2+ in the particulate fraction were very much lower than those in the original homogenate. The addition of the soluble fraction resulted in a marked enhancement of the particulate-bound cyclase activity, while the adenylate cyclase [EC 4.6.1.1] activity was not enhanced. The enhancement was dependent on Ca2+, and the activating factor is suggested to be a protein.


Purification and some properties of two NADP+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1. Two isozymes of NADP+-specific isocitrate dehydrogenase [ICDH; EC 1.1.1.42] were confirmed to be present in an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1, on the basis of the temperature-activity curve and electrophoretic mobilities. These isozymes were separated and purified about 170-fold for isozyme I (specific activity at 40 degrees C, 24.3 units/mg protein) and about 180-fold for isozyme II (specific activity at 20 degrees C, 59.2 units/mg protein), though the isozymes were still not homogeneous. The molecular weights of these isozymes determined by gel filtration were both about 85,000, but the properties of the isozymes were considerably different from each other. The thermostability of isozyme I resembled those of mesophiles, but isozyme II was extremely labile above 20 degrees C. NaCl affected the ICDH isozymes in different ways; the salt protected isozyme I from heat inactivation, but not isozyme II. Nevertheless it enormously enhanced the activity of isozyme II at low concentrations. Moreover, these ICDH isozymes showed different pH optima, Km values for isocitrate, susceptibilities to concerted inhibition by glyoxylate plus oxalacetate, and effects of 2-mercaptoethanol on their stabilities.


Effect of dietary protein content on the activity of rat liver asparagine synthetase. The activity of rat liver asparagine synthetase [EC 6.3.1.1]increased when animals maintained on 25% protein diet were placed on 15% or 6% protein diet. The enzyme activity level rose within one day, reached a maximum in 7 or 10 days after switching the diet and thereafter dropped gradually. During the purification of the enzyme from rats on 25% or 6% protein diet, the yield and increase of the specific activity were similar in the two groups. Combination of the liver extracts from two such groups demonstrated that the amount of endogeneous inhibitors of the enzyme did not change on replacing the diet. The elevation of the enzyme activity in rats fed 6% casein diet was suppressed by an injection of cycloheximide or actinomycin D. It is suggested that the change in the enzyme activity was due to alteration of the amount of the enzyme.


Cross-linking with diimidates of glutamine synthetase from Bacillus stearothermophilus. Glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus was modified with diethyl malonimidate (DEM), dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). DMA modified most epsilon-amino groups. On modification with DMA, formation of 3 to 4 cross-links/subunit resulted in a large increase in thermostability. The activity, allosteric properties and fluorescence spectrum of the enzyme were not changed on cross-linking. The SDS-polyacrylamide gel electrophoretic profiles of DEM-, DMA-, and DMS-modified enzymes suggested that the interaction berween six subunits in each of the two hexagonal rings of the protein are heterologous and are different from those between the piled subunits on different rings.


The oxidation-reduction potentials of compound I/compound II and compound II/ferric couples of horseradish peroxidases A2 and C. The reversibility of the stepwise reduction of Compound I to the ferric state via Compound II was confirmed in horseradish peroxidases A2 and C. The values of E'o (compound I/Compound II) and E'O (Compound II/ferric) were measured from equilibrium data coupled with the K2IrCl6-K3IrCl6 system in a narrow region of pH near 6.3. The ferric enzymes were also oxidized by ferricyanide to Compound II at alkaline pH and the values of E'O (Compound II/ferric) were measured from the equilibrium data. The pH dependence of E'O (Compound II/ferric) was in accord with the equation: E'O = EO + 0.058 log (Kr[H+] + [H+]2)/(KO + [H+]), where Kr and KO are proton dissociation constants in the ferric enzyme and Compound II, respectively. The pH-E'O (Compound I/Compound II) curves were likewise obtained from the equation, E'O = EO + 0.058 log (Kr + [H+]), where Kr is the proton dissociation constant in Compound II. The forward and backward rate constants were measured in each of one-electron transfer reactions of the peroxidases with the K2IrCl6-K3IrCl6 system at various pH values. The E'O values calculated on the assumption that the ratio of the rate constants equals the equilibrium constant were compared with those obtained from the equilibrium data.


Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone. Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.


Metabolic complications of acute arterial occlusions. Metabolic complications secondary to acute arterial occlusions occurred in 7.5% of our cases. It is pointed out that the characteristic biochemical changes: 1) may already be detectable during the ischemic phase, and 2) are more pronounced after revascularization of the extremity. Their identification at the early stage is essential for preventing the serious outcome of the late stage. Amputation rates are quite high (40-50%) and mortality rates are similarly great (30-80%). The ischemic rhabdomyolysis which leads to the clinical and manifestations and biochemical alterations is the initiating pathogenic factor of this syndrome. Prophylaxis and management of these complications were reviewed.


Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium. Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.


Rapid isolation of metaphase chromosomes containing high molecular weight DNA. Metaphase chromosomes with high molecular weight DNA were isolated from Chinese hamster ovary (CHO) cells in a neutral buffer containing polyamines and chelators. The individual, unfixed chromosomes retained their centromeric and secondary constrictions, distinct sister chromatids, and complex banding patterns. The DNA from these chromosomes was 100-fold larger (2 x 10(8) daltons) than DNA from chromosomes isolated by other procedures. These characteristics indicate preservation during isolation of considerable native structure. In contrast to chromosomes produced by other methods, these chromosomes were stable in storage and did not aggregate, thus providing useful material for studies of the structure and biochemistry of individual chromosomes.


Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo. Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.


Immunological studies of subacute measles encephalitis in ferrets: similarities to human subacute sclerosing panencephalitis. Ferrets inoculated with subacute sclerosing panencephalitis virus strains D.R. and Biken developed a subacute encephalitis. Brain extracts, at neutral pH, from these ferrets showed high measles antibody titers, increased concentrations of immunoglobulin G (IgG), and higher IgG/albumin ratios than those of controls. Although the brain extracts of subacute encephalitic animals showed significant synthesis of measles-specific IgG (20 to 60% of the total IgG) within the central nervous system, the electrophoretic patterns of these extracts did not show oligoclonal bands in the gamma-globulin region. Brain residues from most ferrets with subacute encephalitis, when eluted at low pH, demonstrated the presence of bound measles-specific antibodies. Excluding the electrophoresis data, other results are identical to those seen in human subacute sclerosing panencephalitis, indicating that the subacute encephalitis in ferrets may serve as a model for human subacute sclerosing panencephalitis.


Development of dengue virus plaques under serum-free overlay medium. An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.


Development of a chemically defined liquid medium for growth of Legionella pneumophila. A chemically defined liquid medium has been developed for the study of the physiology and antigen production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, alpha-ketoglutaric acid, pyruvate, 0.05% l-cysteine, 0.05% glutathione, and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium in shake culture at 37 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum cell surface antigen as distinguished by specific fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2, pyruvate, and alpha-ketoglutarate strongly stimulated growth; that cysteine and methionine were required for growth; and that serine, threonine, histidine, tyrosine, and tryptophane were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not use glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate, and ethylenediaminetetraacetic acid totally inhibited growth. Beta-mercaptoethanol, thioglycolate, dithiothreitol, and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and antigen production.


Improved procedure for transport of dental plaque samples and other clinical specimens containing anaerobic bacteria. An improved transport system for samples containing anaerobic bacteria was developed. This system increased the recovery rate of anaerobic bacteria up to 28.8% as compared to a commonly used method.


Proteolytic enzymes and plasminogen activator in melanoma. Proteolytic activities were measured in extracts of human skin melanoma, lymphatic metastasis and in nonmalignant naevi by using various proteinase substrates as well as plasminogen activator assay. pH-optima for hydrolysis of various proteinase substrates by these tumors were found to be essentially the same as in healthy human skin. Melanoma extracts were found to especially readily hydrolyze N-alpha-benzoyl-DL-arginine beta-naphthylamine (BANA) at pH 5.8 in the presence of 1 mmol/l dithiothreitol and EDTA (cathepsin B1-like enzyme) as well as histones and p-tosyl-L-arginine methyl ester (TAME) at pH 7.5, and showed increased capacity to activate plasminogen when compared to nonmalignant naevus. The possible role of proteinases in malignant melanoma is discussed.


Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine. The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.


[The action of Buphenin on uteroplacental haemodynamics (author's transl)]. The action of buphenin, a uterine relaxant from the group of adrenergic beta-mimetics, was examined on the blood pools of placenta, myometrium and heart, in 10 pregnant women. The method consisted of an i.v. injection of the radioisotope 113m-Indium and of registering the blood pools by external measurement of the gamma-activity. buphenin caused an increase in placental and myometrial blood pools with a corresponding decrease in cardiac blood pool. These changes were observed in all the cases examined and occurred whether the uterus had previously been inactive or in labour. The changes in blood pool were dependent on the dose of buphenin administered but there was no relationship apparent between the degree of pool changes and severity of pregnancy complication prevailing.


Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system. A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.


Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats. In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.


Distribution of glutamine synthetase in the rat central nervous system. The results of a light microscopic immunohistochemical study of glutamine synthetase in rat nervous system are presented. In all sites studied the enzyme was confined to astrocytes. Except for trace amounts in ependymal cells, the enzyme was not observed in other cells of the nervous system including neurons, choroid plexus, third ventricular tanycytes, subependymal cells and mesodermally-derived elements. The intensity of astrocyte staining varied in different regions with the greatest degree noted in the hippocampus and cerebellar cortex while the least was noted in brain stem, deep cerebellar nuclei and spinal cord. The glutamine synthetase content correlated well with sites of suspected glutamergic activity in keeping with the view of a critical role of astrocytes in the regulation of the putative neurotransmitter glutamic acid.


Cytochemical localization of alkaline phosphatase and Na+-pump sites in adult rat colon. The cellular and subcellular locialization of alkaline and K+-dependent phosphatase activities in the colonic mucosa of adult rats and rabbits was studied with the electron microscope. The 1-cysteine-sensitive alkaline phosphatase activity was observed in the brush border membrane of the chief cells. The contraluminal plasma membrane of chief cells was devoid of this enzyme activity. In contrast, the cardiac glycoside-sensitive K+-dependent phosphatase was predominantly localized in this region of the cheif cells.


Adoptive immunotherapy of leukemia in the rat, without graft-VS-host complications. PVG rats bearing a transplantable T cell leukemia were treated with large inocula of lymphoid cells from AUG rats sensitized either against the leukemia or against PVG lymphocytes. AUG and PVG bear identical Ag-B antigens but differ at minor loci, including the Pta loci, which code for differentiation antigens expressed only on peripheral T lymphocytes. Treatment with AUG cells immune to either the PVG leukemia or normal PVG cells resulted in prolonged survival of leukemic rats, a profound but ephemeral leukopenia and prolonged disappearance of leukemic cells from lymphoid tissue. All treated animals, however, eventually died with large, discrete deposits of leukemic cells in both hard and soft tissues. Despite the deliberate mismatching of host and donor cells for minor transplanation antigens, no evidence of GVH symptoms was observed in treated rats. This was interpreted as a result of directing the adoptive immune response to antigens of restricted distribution, i.e., on leukocytes and not on somatic cells.


Polyglutaraldehyde: a new reagent for coupling proteins to microspheres and for labeling cell-surface receptions. II. Simplified labeling method by means of non-magnetic and magnetic polyglutaraldehyde microspheres. Procedures were developed for the synthesis of a new immunoreagent in form of polyglutaraldehyde (PGL) microspheres in sizes ranging from about 50 nm to 1.5 micron. Addition of fluorochromes during synthesis yielded microspheres of high fluorescence intensity. By carrying out the polymerization of glutaraldehyde in presence of iron oxide, magnetic PGL microspheres were produced. Antibody conjugates obtained by interaction of PGL microspheres with immunoglobulins were used to label human red blood cells (RBC) and lymphocytes. A simple method for the separation of magnetically labeled human RBC from unlabeled cells was demonstrated.


Production of maximally acid urine by the isolated dog kidney. The isolated kidney has not been reported to acidify urine maximally. To study this defect, kidneys from dogs fed NH4Cl were perfused with autologous blood. Perfusate pH was 7.20 +/- 0.03 [HCO3] was 14 +/- 1 mEq/L, and urine pH was abnormally high, 6.60 +/- 0.08. When corrected for difference in GFR, UNH4+V was similar to that seen in vivo, but UTAV and UNet H+V were low. FEHCO3- was 2.3% +/- 0.8% and HCO3- excretion persisted to a small degree at perfusate [HCO3-] of 8 to 9 mEq/L. In response to HCO3- infusions, large increases in excretion were not seen until perfusate values were over 24 to 26 mEq/L. HCO3- Tmax was 2.94 +/- 0.07 mEq/dl of glomerular filtrate. The isolated kidney failed to raise U-B PCO2 with HCO3- infusion secondary to low urine [HCO3-] and [Pi]. During perfusion in another group of kidneys from dogs fed NH4Cl and given DOC, perfusate pH and [HCO3-] were similar to those in the first group. Urine pH was also inappropriately high, 7.12 +/- 0.09, and there was no UNet H+V. In response to Na2SO4 infusion, urinary pH fell to 5.00 +/- 0.27. Log10UUAV was correlated to urine pH during the control perfusions in both groups and after Na2SO4 in the NH4Cl + DOC group. Thus production of a low urine pH in the isolated kidney may be mediated by changes in transtubular potential difference resulting from increased distal nephron delivery of Na+ and nonabsorbable anion. The defect in acidification is similar to that observed in incomplete forms of clinical type 1 (distal) renal tubular acidosis.


Depletion of red cell ATP by incubation with 2-deoxyglucose. In order to devise a more physiologic system for measuring depletion of red cell ATP levels, the effect of incubating human erythrocytes with 2-deoxyglucose has been investigated. ATP depletion proceeds very slowly at a 20 mM concentration of 2-deoxyglucose, a level which exceeds the Km of hexokinase for this substrate by more than 10-fold. However, at 160 mM concentration of 2-deoxyglucose, ATP depletion proceeds sufficiently rapidly that nearly 90% of ATP has disappeared from the red cell after 2 1/2 hr of incubation. To explain this observation, a number of additional studies were carried out. It was found that 2-deoxyglucose penetrated rapidly into red cells. Phosphorylation of 2-deoxyglucose in red cells was inhibited by both products of the 2-deoxyglucose-phosphorylating reaction, namely, 2-deoxyglucose-6-phosphate and ADP. Inhibition of 2-deoxyglucose phosphorylation was diminished at higher-than-physiologic pH levels. Red cells may be relatively rapidly depleted of ATP by incubation with 100 mM 2-deoxyglucose in a saline-phosphate-buffered medium, pH 7.8. In such rapidly depleted cells, the morphologic changes which formerly were attributed to ATP depletion do not occur.


The fate of fetal and adult B-cell progenitors grafted into immunodeficient CBA/N mice. The relative ability of various precursors to generate functional B cells in vivo was assessed by transferring normal, chromosomally-marked CBA/H-T6T6 cells to irradiated or unirradiated immunodeficient CBA/N mice. Emergence of donor-derived B cells was monitored by means of a B-cell cloning assay (in which CBA/N cells are inactive), and by karyotpic analysis of lymphoid, myeloid, and stem cell metaphases. Grafts of lymph node, spleen, anti-mu surface immunoglobin suppressed bone marrow, sIg+ cell-depleted marrow, normal marrow, fetal liver, and yolk sac suggest: (a) there is little self-renewal of sIg+ B cells in these models; (b) pre-committed cells have extensive proliferative/differentiative potential and at least initially contribute most of the newly-formed B cells; (c) populations or pre-B cells obtained from various sources differ in their regenerative ability; (d) CBA/N mice are deficient in a category of pre-B cells which are found in fetal liver; and (e) selective B-cell chimerism results from grafting of unirradiated CBA/N mice.


Generation of anti-type III pneumococcal polysaccharide hybridomas from mice with an X-linked B-lymphocyte defect. (CBA/N X BALB/c male)F1 mice bear on X-linked defect making them totally unresponsive to T-independent (TI), TI-2 antigens such as type III pneumococcal polysaccharide (SSS-III). We found that somatic cell hybrids between CB nonresponder spleen cells and NS1 plasmacytoma cells secreted antibody specific for SSS-III. The solid-phase binding of such antibody was completely inhibited by the addition of free antigen (SSS-III) and the amount of antibody detected in culture fluids ranged from 10 ng/ml to 10 micrograms/ml. Eight hybridoma clones were identified; all make antibody of the IgM class. These results indicate that the X-linked defect does not result in a deletion of a B-cell subset which responds to TI-2 antigens.


Primary affective disorders.

Primary affective disorders. This paper reviews the diagnosis and medical treatment of the major affective disorders. Patients with severe mood disturbances are frequently seen by the family physician. The diagnosis may be delayed since the patient may focus predominantly on somatic concerns which may mimic physical illness. The characteristics, course, and differential diagnosis of depression and mania are discussed. Antidepressants and lithium therapy greatly improve the prognosis of these disorders; monoamine oxidase inhibitors and neuroleptics are indicated for special subtypes of depression. Dosage schedules, interactional effects, adverse and toxic effects are reviewed for tricyclic antidepressants and lithium.


Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants. The purpose of these experiments is to test whether the differences between normal and tetrodotoxin-resistant Na+ channels reside in the selectivity filter. To do this, we have compared the selectivity of batrachotoxin-activated channels for alkali cations, organic cations, and nonelectrolytes in two neuroblastoma clonal cell lines: N18, which has normal tetrodotoxin (TTX) sensitivity, and C9, which is relatively TTX-resistant. We have also studied the effect of H+ on Na+ permeability and on the interaction between TTX and its receptor site in both cell lines. There is no qualitative difference between the two cell lines in any of these properties. In both cell lines the batrachotoxin-activated Na+ channels have a selectivity sequence of Tl+ greater than Na+ greater than K+, guanidinium greater than Rb+ greater than Cs+, methylamine. Also, in both cell lines H+ blocks Na+ channels with a pKa of 5.5 and inhibits the action of TTX with the same pKa. These observations indicate that the selectivity filters of the Na+ channels in C9 and N18 do not differ significantly despite the 100-fold difference in TTX-affinity. Our selectivity studies of batrachotoxin-activated Na+ channels for both cell lines suggest that these toxin-activated Na+ channels have a limiting pore size of 3.8 x 6.0 A, as compared to a pore size of 3.0 x 5.0 A for potential-activated Na+ channels.


Gustatory responses of eel palatine receptors to amino acids and carboxylic acids. The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.


Glutamine synthetase and glutamate synthase activities during growth and sporulation in Bacillus subtilis. Glutamine synthetase in Bacillus subtilis 168 was repressed to a greater extent by L-glutamine or L-arginine than by ammonia when each was used as sole nitrogen source. It was derepressed when either L-glutamate or nitrate was used as nitrogen source. Glutamate synthase was repressed by  L-glutamate or L-arginine and, to a lesser extent, by L-glutamine but was derepressed during growth with ammonia or nitrate. Glutamine synthetase activity was unaltered during the onset of sporulation. Glutamate synthase activity, however, underwent a small and apparently transient increase in bacteria induced to sporulate by nitrogen limitation.


The effects of temperature and pH variations on plaque production by different virulence types of myxoma virus. Eight 'strains' of myxoma virus, spanning the complete spectrum of virulence, were tested for ability to produce plaques on rabbit kidney cells at varying temperatures and pH values. A positive correlation was found between virulence in rabbits and ability to produce plaques at supra-optimal temperature and at low pH.


Rotavirus stability and inactivation.

Rotavirus stability and inactivation. The stability of the infectivity of Simian rotavirus, SA11, has been analysed and compared to the stability of reovirus type 1. SA11 infectivity was stable to freeze-thawing, sonication, incubation at 25 degrees C overnight or at 37 degrees C for 1 h and to treatment with acid, ether, chloroform and Genetron. In contrast to reovirus, the infectivity of SA11 was more rapidly inactivated by heating at 50 degrees C. SA11 infectivity was inactivated above pH 10.0 and by heating at 50 degrees C in 2 M-MgCl2, but was stabilized by heating in 2 m-MgSO4; reovirus 1 infectivity was enhanced by heating in MgCl2. Both SA11 and reovirus 1 were inactivated by freezing in MgCl2. These results show that rotaviruses and reoviruses can be distinguished by their patterns of inactivation by physical and chemical agents.


Haemolysis by two alphaviruses: Semliki Forest and Sindbis virus. Purified preparations of Semliki Forest (SFV) and Sindbis virus haemolyse red blood cells from several species of animals and birds. The optimal haemolysis by SFV was obtained at pH 5.8 with 1-day-old chick erythrocytes incubated at room temperature. Considerable variation in haemolytic activity was observed between different virus preparations purified by different methods. The haemolytic activity of SFV was inhibited by antisera against whole virus or isolated envelope proteins but not with antiserum against virus capsid protein. Neither lipid and detergent-free envelope protein octamers with high haemaggluinating titre, nor isolated nucleocapsids caused haemolysis. Fresh, unpurified SFV and Sindbis virus preparations did not haemolyse unless they were exposed for repeated cycles of freezing and thawing. It appears that the haemolytic activity resides in the virus glycoproteins(s) but can only be manifested in slightly damaged whole virus particles.


Investigation of the system cobalt(II) bovine carbonic anhydrase B-trichloroacetaldehyde. The interactions between hydrated trichloroacetaldehyde and cobalt(II)bovine carbonic anhydrase B have been investigated as a function of pH by means of electronic spectroscopy of FT nmr spectroscopy. The hydrated aldehyde is bound to the metal ion and its apparent affinity constant is pH dependent with a bell-shaped profile. The kinetic parameters of the dissociation process have also been determined.


Multiple, prolonged actions of neuroendocrine bag cells on neurons in Aplysia. I. Effects on bursting pacemaker neurons. 1. The bag cells are a group of neuroendocrine cells located in the abdominal ganglion of Aplysia. Accumulated evidence suggests they synthesize and release egg-laying hormone (ELH), a peptide that induces egg laying. In this and the following paper (37) we describe five types of prolonged neural responses in cells of the isolated abdominal ganglion that are produced by stimulated bag cell activity. 2. Prolonged, 5- to 40-min bursts of spike activity were triggered in the normally silent bag cells by local stimulation of one of the bag cell clusters with brief, 0.6- to 2-strains of pulses. This local stimulation minimized the possible effects of the stimulus on other ganglion cells and initiated bag cell activity similar to what has been recorded in intact animals at the initiation of egg laying. 3. Following onset of triggered bag cell activity there is an increase in the amplitude of the bursting pacemaker potential in cell R15 that results in augmented bursting activity in this autoactive cell for up to 3 h. The increase begins in less than 1 min and reaches a maximim after 8--20 min. In two other bursting pacemaker cells, L3 and L6, there is a second type of response, slow inhibition, consisting of a smoothly graded hyperpolarization that begins in 5--14 s, reaches a peak value of 10--20 mV after 30 s, and results in a decrease in the spontaneous spike activity of these cells for 3 h or longer. Both types of responses are contingent on the occurrence of bag cell activity, they depend on prolonged bag cell activity for their normal expression, and they occur in the absence of the fast interactions characteristic of conventional synapses. 4. The results reveal at the level of intracellular recordings prolonged actions of peptide-secreting neuroendocrine cells on the central nervous system. The role of ELH as a putative mediator of one or more of these actions is discussed.


Neutral lipid accumulation in yeast due to inositol deficiency: kinetic studies on the reciprocal regulation by fructose bisphosphate and citrate of yeast acetyl CoA carboxylase. Neutral lipids, especially triacylglycerols, accumulated due to myo-inositol deficiency both in the cells of Saccharomyces carlsbergensis (Hayashi et al. (1976) J. Biol. Chem., 251, 5759--5769) and in the liver of the rat (Hayashi et al. (1974) Biochim. Biophys. Acta, 360, 134--155). The accumulation of triacylglycerols in the deficient yeast resulted, at least partly, from an enhancement of acetyl CoA carboxylase activity. The activation of the enzyme reflected the fluctuation due to the deficiency in the levels of fructose bisphosphate and citrate (Hayashi et al. (1978) Biochim. Biophys. Acta, 540, 231--237). Thus, the kinetics of the regulation of acetyl CoA carboxylase by these intermediates was studied. In physiological concentrations fructose bisphosphate sigmoidally activated acetyl CoA carboxylase from yeast with the Hill coefficient of 3, while citrate counteracted the fructose bisphosphate activation in a sigmoidal manner with the Hill coefficient of 2. Fructose bisphosphate markedly increased the apparent Vmax value of acetyl CoA carboxylase for the substrate, ATP and slightly decreased the apparent Km value. Citrate greatly decreased the apparent Vmax value increased by fructose bisphosphate.


Sulfapyridine metabolites in children with inflammatory bowel disease receiving sulfasalazine. Fifteen children followed as outpatients with chronic inflammatory disease of the colon were given sulfasalazine in doses from 1 to 4 gm/day (22 to 68 mg/kg, or 0.69 to 2.33 gm/m2). No correlation was found between the dose/m2 administered and the total serum sulfapyridine levels. However, 11 of 15 patients achieved SP levels greater than or equal to 17 micrograms/ml, a level approximating that reputedly associated with therapeutic efficacy. Patients who were either slow acetylators or slow hydroxylators of sulfapyridine had total SP levels significantly higher than patients who were both rapid acetylators and hydroxylators (20.0 +/- 1.2 vs 14.6 +/- 1.6). Total SP serum levels were not correlated with the activity of the disease. No toxic levels (greater than 50 micrograms/ml of SP) were encountered. We conclude that a dose of SASP in the range of 1.5 to 2.0 gm/m2 can be safely administered to children and is usually associated with serum SP levels considered in the therapeutic range. Although one-third of children are both rapid acetylators and hydroxylators and will have somewhat lower SP levels, the routine monitoring of SASP therapy with SP levels is not necessary for management of disease.


Rapid, large-scale isolation of Plasmodium berghei sporozoites from infected mosquitoes. The discontinuous gradient technique for recovery of malarial sporozoites from mosquitoes (Beaudoin et al., 1977) has been modified to speed up recovery and prevent sensitization of mice by components of the gradient which contaminate the sporozoites used as antigen. Mouse serum was substituted for BSA in the gradient because the latter produced hypersensitivity. Best results were obtained with gradients consisting of Medium 199, Renografin and mouse serum. Heavy and light solution of gradient components are layered in a centrifuge tube. Centrifugation of comminuted, infected mosquitoes applied to the top of the discontinuous gradient concentrates sporozoites at the interface. Sporozoites recovered from the gradient were infective, immunogenic, and relatively free of mosquito tissue. This improved method enables recovery of 100,000 sporozoites from each Anopheles stephensi infected with the ANKA strain of Plasmodium berghei. As many as 2,800 mosquitoes have been processed in 2 hr without a significant decrease in yield.


Heterotopic splenic autotransplantation in prevention of overwhelming postsplenectomy infection. An experimental study was undertaken to evaluate the protective effect of heterotopic splenic autotransplantation in weanling rats. Rats were divided into three experimental groups: splenectomy, control, and splenic autotransplantation. Rats were challenged with i.v. type I pneumococcus. Bacterial bloodstream clearance and survival were determined. Splenic bacterial uptake was measured by determining the isotopic activity of technetium-99m-labeled pneumococci. Autoradiographs and material stained with hematoxylin and eosin and Gram strains were examined for histologic features. All autografts survived and were histologically compatible with normal splenic tissue. Bloodstream clearance of pneumococci was significantly greater in rats with splenic autotransplantation. Splenic autografts had 10 to 30 times greater uptake of pneumocci than did the liver. Rats with autotransplantation had a prolonged survival time. Heterotopic splenic autotransplantation may prove to be an important adjunctive surgical measure in the treatment of children undergoing splenectomy.


The distribution of amylobarbitone, butobarbitone, pentobarbitone and quinalbarbitone and the hydroxylated metabolites in man. Fluid and tissue specimens collected from 30 subjects at autopsy have been assayed for their content of common sedative barbiturates and the corresponding hydroxylated metabolites by g.l.c. Where one barbiturate had been ingested an inverse relationship between lipid solubility of the drug and the distribution in fluids and tissues was observed. In most cases the liver, and in the remainder the spleen, contained the highest concentrations of barbiturate. Bile concentrations were often in excess of those in the corresponding liver. The metabolites of the four sedative barbiturates were usually present in lower amounts than the parent drugs in the fluids and tissues of most subjects but urine often contained much higher concentrations of metabolites--sometimes exceeding that of the parent drug in the liver. Administration of two or more barbiturates together did not appear to affect the distribution and metabolism of the individual drugs.


Effects of the non-steroidal anti-inflammatory drug benoxaprofen on leucocyte migration. Possible modes of action of the anti-inflammatory drug, benoxaprofen, have been explored. The drug caused inhibition of leucocyte migration, principally of mononuclear cells into the pleural cavity of rats undergoing carrageenan-induced pleurisy. Evidence was obtained from in vitro leucocyte migration and chemotaxis models that the drug acted directly on the mononuclear cells rather than by inhibition of chemotactic factors.


N-Chloroacetyl 5-methoxytryptamine (isamide): a selective antagonist of 5-hydroxytryptamine in the rat uterus. Isamide, the N-chloroacetyl derivative of 5-methoxytryptamine, produced a dose-dependent competitive blockade of uterine contractions in vitro induced by 5-HT. The pA2 value for the 5-HT-isamide interaction was 4.42. The blockade was short-lasting and reversible; after recovery, a dose-dependent increase in the uterine sensitivity to 5-HT was found. The blockade proved to be selective to the 5-HT receptor. The simultaneous application of 5-HT plus isamide partially prevented the 5-HT-induced auto blockade phenomenon. In addition, isamide did not affect the contractile responses of the uterus to oxytocin or bradykinin or the contractile effects of the rat vas deferens to adrenaline.


Specific membrane-binding of angiotensin II--the role of degradative enzymes. The possible role of degradative enzymes was examined in the specific binding of angiotensin II (AII) to cell membranes. Red blood cell membranes did not bind AII specifically under any of the ambient conditions studied, indicating a lack of AII receptors and no role for the degradative enzymes in specific binding. Rabbit aorta smooth muscle cell membranes bound AII specifically, and this binding had similar characteristics to those previously described for this preparation. It is concluded that specific binding of AII to cell membranes does not involve degradative enzymes, and probably represents binding to the biologically active receptor.


Rapid paper chromatographic separation of [14C] angiotensen II from some metabolites: application to organ distribution. A rapid, inexpensive method for the separation of 5-1-isoleucyl[14C] angiotensin II (A-II) from its various metabolites has been devised. A-II was extracted from tissues with absolute methanol (recovery 96%) and paper chromatographed in a butanol-acetic acid-water (18:2:5) medium for two ascents at 60 degrees C. The resulting RF for A-II of 0.45 was then compared with the RF values of three A-II metabolites produced by enzymatic degradation of the 14C-A-II and [14C]isoleucine. Trypsin degradation produced the [14C]hexapeptide metabolite, chymotryptic degradation produced the [14C]tetrapeptide metabolite and carboxypeptidase A degradation produced the [14C]heptapeptide. Increases in temperature produced a continuous increase in RF values for all the substances examined but the resolution decreased above 60 degrees C. Similarly, increases in the temperature caused the appearance of secondary peaks with some but not all peptides. The tryptic digest (hexapeptide) and the chymotryptic digest (tetrapeptide) are apparently acid- and heat-stable under the experimental conditions. All of the peptides examined failed to produce secondary peaks when heated at neutral pH. The method was used to study the tissue distribution of 14C-A-II after intravenous injection.


Synthesis, properties and biological activity of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide -- a new ligand for epithelial sodium channels. A method is described for the synthesis and purification of tritiated N-benzylamidino-3,5-diamino-6-chloro-pyrazine carboxamide (benzamil). The tritium was inserted at the meta position of the benzyl ring, from which it apparently does not exchange with solvent hydrogen. When stored in ethanol at -4 degrees C the radioligand remains stable for at least 15 months. The pharmacology of benzamil is very similar to that of amiloride in terms of its effects on sodium transporting epithelia except that it has a higher affinity. The affinity of benzamil for sodium channels in amphibian epithelia in the absence of sodium is approximately 10(9) M-1. The new ligand can be used to label sodium channels in epithelia, and may be useful in channel isolation procedures.


A nuclear magnetic relaxation study of bound water in solutions of disodium cromoglycate. The amount of water bound to disodium cromoglycate (DSCG) in frozen aqueous solutions was determined from the amplitude of the n.m.r. free induction decay. The hydration number was found to be 15 molecules H2O/molecule DSCG of which 6 H2O molecules were less strongly bound than the rest. Proton T1 and T2 relaxation times in non-frozen solutions implied a values of 10(-8) s for the correlation time of the bound water at 291 K. This is consistent with the mobility of DSCG molecules in a smectic mesophase.


The surface activity and self-association of some beta-adrenoceptor blocking agents in aqueous solution. The surface activity at the air-solution interface of a series of beta-adrenoceptor blocking agents has been determined. The drugs investigated included, propranolol, sotalol, oxprenolol, labetolol, timolol, metoprolol and acebutolol. Correlation between surface activity and local anaesthetic potency of the drugs is examined. Light scattering measurements have indicated self-association of sotalol, oxprenolol, acebutolol and metoprolol in 0.5 mol kg-1 sodium chloride. Critical micelle concentrations and aggregation numbers are reported.


Placebo granules as cores for timed release drug delivery systems. A drug delivery system is proposed constituted of spherical placebo granules as cores with polymeric surface films containing drug. This timed release dosage form has been prepared by means of a fluidized bed coating technique using ethyl cellulose as the polymeric film and caffeine and salicylic acid as model drugs. The release of the drugs from the dosage form (a) at different drug concentrations and (b) into solutions of different pH showed that drug release was linearly related to the square root of time. Good agreement was found between the theoretical release rate of caffeine, calculated according to Higuchi's equation for a homogenous matrix using membrane permeation parameters measured on linear films, and the experimental results in the case of low drug concentrations. Deviation of the release rate from the homogenous model at high drug concentrations could be explained by crystallization of the drug from the film.


The stability of betamethasone-17-valerate in semi-solid bases. The stability of betamethasone-17-valerate in semi-solid bases has been investigated. Emphasis has been placed on the stability problems which could arise upon dilution of proprietary preparations by the use of model systems. Betamethasone-17-valerate has been shown to decompose to betamethasone-21-valerate and betamethasone alcohol. Quantitation of the decomposition was by direct densitometry on thin layer chromatographic plates. The decomposition was found to be an apparent first order process and to depend on the diluent used and its concentration. Attempts were also made to relate the rate of decomposition to the pH of the base used, and to stabilize the products.


The determination of aluminium in antacid formulations by Gran's Plot titration with sodium fluoride. The aluminium content of a number of proprietary antacid formulations has been determined by titration with sodium fluoride, using the fluoride activity electrode as an end-point detector. Results compared favourably with the official method. The fluoride titration, however, involved less sample manipulation and was more rapid to carry out. End-point detection was made easier by treating titration data by the Gran's Plot method.


Ionization kinetics of the carbon acid phenindione. The ionization kinetics of carbon acids are slow relative to those of classical acids and bases. Phenindione (2-phenyl-1,3-indandione) is a 1,3-diketone carbon acid of macroscopic experimental pKa 4.09 at 25 degrees and ionic strength 0.1. The ionization kinetics of phenindione were determined at an ionic strength of 0.1 and 25 degrees using stopped-flow spectrophotometry and a pH jump technique. A log k'obs-pH profile for the approach to the ionization equilibrium was determined, and a mechanism consistent with the profile was postulated. The percent enol versus diketo form of phenindione and the pKaenol and pKadiketo were calculated from the kinetic data. Phenindione acid deprotonation kinetics by various oxygen and nitrogen bases suggested that, with bases of pKa 4.7--8.5 and in the pH 5--8.5 range, the acidic phenindione proton as approximately 45% transferred in the rate-determining transition state.


Kinetics and mechanism of ionization of the carbon acids 4'-substituted 2-phenyl-1,3-indandiones. The ionization kinetics of 1.3-diketone carbon acids are slow relative to those of classical acids and bases. The ionization kinetics of three 4'-substituted 2-phenyl-1,3-indandiones, 4'-chloro-, 4'-methoxy-, and 2-phenyl-1,3-indandione itself, were studied at 25 degrees and ionic strength 0.1 using stopped-flow spectrometry and a pH jump technique. A log k'obs-pH profile for the approach to the ionization equilibrium was consistent with a reaction scheme postulated earlier for the ionization of another carbon acid, phenylbutazone. The percent enol versus diketo form of the acids and the pKaenol and pKadiketo were calculated from the kinetic data. Hammett plots of the various kinetic and equilibrium constants supported a mechanism for acid deprotonation consistent with proton abstraction being the predominant process when very weak bases such as water were the proton acceptors. Desolvation effects and the work required to get the two reacting molecules together in the correct configurations predominated when the proton abstraction was by stronger proton acceptors.


Differential potentiometric method for determining dissociation constants of very slightly water-soluble drugs applied to the sulfonamide diuretic chlorthalidone. A renewed application of potentiometric acid-base titrations is described, by which dissociation constants of practically water-insoluble drugs can be measured accurately. The method uses the difference in the amount of titrant between a suitable aqueous solvent and a solution of the drug in that solvent. Such potentiometric difference titrations were conducted on a 3.7 X 10(-4) M solution of chlorthalidone in 0.1 M aqueous KCl in the pH 3.5--10.6 range at 25 degrees. Nonlinear least-squares regression analysis was applied to the data. From four determinations, a value of 9.24 +/- 0.02 (mean +/- SEM) resulted for the apparent dissociation constant of the first chlorthalidone acid group. The thermodynamic dissociation constant was calculated at pKa1 = 9.35 (25 degrees) by using a correction for activity.


Kinetics and mechanism of chlorambucil hydrolysis. The kinetics and mechanism of hydrolysis of the cytotoxic drug chlorambucil were investigated. A study of the reaction order and of the pH, dielectric constant, and ionic strength effects on the reaction rate revealed that the hydrolysis was a limiting unimolecular nucleophilic substitution, the rate-determining step being the ionization of the first 2-chloroethylamine chlorine. The hydrolysis rate was markedly dependent on the protonation degree of the chlorambucil basic group. indicating that a cyclic ethyleneimmonium ion may be involved in the rate-determining step.


Comparison of the effects of the isomers of amphetamine, methylphenidate and deoxypipradrol on the uptake of l-[3H]norepinephrine and [3H]dopamine by synaptic vesicles from rat whole brain, striatum and hypothalamus. The ATP-Mg++-dependent uptake of [3H]dopamine and l-[3H]norepinephrine into purified synaptic vesicles of whole rat brain, rat striatum and rat hypothalamus was inhibited 10-fold more effectively by S-(+)-amphetamine as compared to its corresponding (R-(-)-enantiomer. In contrast, S-(+)-deoxypipradrol and its R-(-)-enantiomer were approximately equipotent inhibitors of 3H-amine uptake into these synaptic vesicular preparations. The 1R:2R-methylphenidate was twice as potent as its 1R:2S-enantiomer as an inhibitor of 3H-catecholamine uptake. These data suggest that the receptor sites on the amine pumps present in the membranes of all three vesicular preparations are similar in so far as they are all sensitive to the stereochemical configuration around the alpha-carbon of amphetamine but are not sensitive to the stereochemical configuration around the analogous carbon of deoxypipradrol and methylphenidate. These observations are the reverse of those previously observed for the phenethylamine pumps present in peripheral and central neuronal membranes.


Agonist-induced changes in beta adrenergic receptor density and receptor-mediated responsiveness in slices of rat cerebral cortex. Incubation of slices of rat cerebral cortex with the beta adrenergic receptor agonist (-)-isoproterenol led to a 30 to 50% decrease in the number of binding sites for [125I]iodohydroxybenzylpindolol and to a 60 to 80% decrease in isoproterenol-stimulated cyclic AMP accumulation. The density of beta adrenergic receptors was also decreased following incubation with (-)-norepinephrine but not with (+)-isoproterenol or dopamine and the decrease in receptor density was blocked by co-incubation with the beta adrenergic receptor antagonist sotalol. The half-time for loss of receptors was approximately 3 min and recovery was observed during a 1 hr reincubation of tissue slices or following exposure to guanine nucleotides. A decrease in beta adrenergic receptor density was also observed following chronic treatment with desmethylimipramine which blocks norepinephrine reuptake and thus potentiates the effects of neurally released norepinephrine at adrenergic receptors. The loss of receptors induced in vitro could be reversed by reincubation or by exposure to guanine nucleotides. In contrast, the loss of receptors induced in vivo was not affected by these procedures.


Pancreatic acinar cells: the effect of carbon dioxide, ammonium chloride and acetylcholine on intercellular communication. 1. Segments of mouse pancreatic or exorbital lacrimal gland were superfused with saline solutions. Under visual control two micro-electrodes were inserted into neighbouring cells within the same acinus or into neighbouring acini. Cell to cell electrical coupling was assessed by injecting rectangular current pulses through one electrode and measuring the electrotonic potential change in the same cell (V(1)) and in the neighbouring cell (V(2)). Acetylcholine (ACh) was added locally to impaled acini by micro-ionophoresis from an extracellular micropipette.2. Exposure of the tissues to a Krebs solution equilibrated with 100% CO(2) caused a rapid increase in the size of electrotonic potential changes in the current injection cell and disappearance of the electrotonic potential changes in a neighbouring acinus or cell. This electrical uncoupling of previously coupled cells was rapidly reversible upon return to a solution equilibrated with 95% O(2) and 5% CO(2).3. Reduction of electrical intercellular coupling was also obtained using smaller CO(2) concentrations (50, 20 or 10%). In these cases the effects developed more slowly and were less dramatic. Reducing the extracellular HCO(3) concentration enhanced the uncoupling effect of 10 or 20% CO(2). However, weak uncoupling effects were still observed using 10 or 20% CO(2) in combination with a high bicarbonate concentration maintaining a constant extracellular pH (7.4).4. Reductions in extracellular pH (down to 5.5) achieved by varying combinations of Tris base and Tris HCl had no effect on electrical coupling. Brief periods of anoxia (100% N(2)) also had no effect.5. Exposure to 20% CO(2) markedly enhanced the uncoupling effect of a brief ionophoretic pulse of ACh.6. Exposure of the tissue to 10 mM-NH(4)Cl, a procedure expected to increase the intracellular pH, counteracted the uncoupling effect of ACh. During sustained uncoupling caused by a sustained ACh stimulation a brief period of exposure to NH(4)Cl caused an immediate and fully reversible recoupling.7. It is concluded that variations in intracellular pH have marked effects on the electrical coupling between neighbouring cells in the pancreatic and lacrimal acinar tissue.


Micro-electrode measurement of skin pH in humans during ischaemia, hypoxia and local hypothermia. 1. The extracellular pH value in the dermis of human skin (skin pH) was measured in vivo using glass micro-electrodes. They were found to be both reliable and accurate. 2. The mean value of skin pH measured in the legs of forty different volunteers was found to be pH 7.54 +/- 0.09 (S.D.). No difference in skin pH was observed between males and females, or in different regions of the limb. 3. Local reductions in skin surface temperature in ten subjects caused an increase of pH 0.023 +/- 0.007 per degree C fall. 4. A 20 min period of tourniquet ischaemia in twenty volunteers induced a fall in skin pH of 0.13 +/- 0.05 units. 5. Hyperventilation during a 10 min period of breathing 10% O2 in N2 caused an increase of pH 0.04 +/- 0.02 in the skin of healthy subjects. 6. Skin pH fell to a value 0.02 +/- 0.02 units below normal 10 min after the hypoxic period, suggesting the presence of excess lactate. 7. Skin pH results compared well with blood gases and pH values of arterialized samples taken during hypoxia. 8. It was concluded that the system was suitable for clinical trials.


Chloride, sodium, potassium and hydrogen ion transport in isolated canine gastric mucosa. 1. The fluxes of isotopically labelled Na+, Cl- and K+ in each direction and H+ secretion across isolated dog gastric mucosa were measured under short-circuit conditions. 2. In the non-stimulated state, the net flux of Na+ was 6.61 micronequiv/cm2.hr from mucosal (luminal, secretory) to serosal (nutrient, blood) side, whereas the net flux of Cl- was only 0.79 micronequiv/cm2.hr, and the direction was from serosal to mucosal side. 3. There was a positive correlation between the net flux of Cl- and acid secretion, however, net flux of Na+ was not correlated with acid secretion initiated by secretagogue treatment. 4. With ion substitution studies, only replacement of mucosal Na+ with choline produced a highly significant decrease in potential difference (p.d.). This indicates that active transport of Na+ from the mucosal to the serosal side is the most important source for the generation of the gastric p.d. in dog gastric mucosa. 5. From ion substitution studies, it was also observed that Cl- in either mucosal or serosal solution is necessary for maintaining acid secretion; whereas only serosal Na+ and K+ are essential for acid secretion. Removal of either Na+ or K+ from the mucosal solution had no effect on acid secretion. 6. Substitution of SO2-(4) for Cl- had no effect on active transport of Na+, but choline substitution for Na+ diminished active transport of Cl-.


In vitro binding of Trypanosoma congolense to erythrocytes. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin-trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.


Partial purification and characterization of a bacteriolytic enzyme secreted by Tetrahymena. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3--4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded 14C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacteriolytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.


Cytoplasmic gel and water relations of axon. A previous method of measuring the swelling pressure (delta IIg) of the cytoplasmic gel of the giant axon of Loligo vulgaris was refined. The estimates of delta IIg made with the improved method were consistent with those made with the earlier method. In these methods the activity of the solvent in the gel is measured by increasing the activity of the solvent in the internal phase of the gel by application of hydrostatic pressure to the gel directly. Comparable values for the activity of the solvent in the gel were obtained also by an alternate method, in which the deswelling of the gel is measured upon decreasing the activity of the solvent in the external phase by addition of a nonpenetrating high mol wt polymer (i.e., Ficoll). Additional support was obtained for the earlier suggestion that delta IIg contributes to the swelling and shrinkage pattern of the whole axon. In part, the new evidence involved two consecutive direct measurements of intraxonal pressure. The first measurement was that of a mixed pressure composed of delta IIg and delta IIm (delta IIm being the effective osmotic pressure due to the intra-extraxonal gradient in the activity of mobile solutes). The subsequent measurement was that of delta IIg alone. The latter measurement was made feasible by destroying the axolemma, thereby eliminating the contribution of delta IIm. An estimate of delta IIm was obtained by subtracting delta IIg from the total pressure measured initially. The delta IIm determined by the above method was two orders of magnitude smaller than the theoretical osmotic pressure. This is consistent with the delta IIm determined previously, where osmotic intra-extraxonal filtration coefficients were compared to the hydrostatic. The mixed pressure experiments lend credence to the idea that the substantial contribution of delta IIg to the water relations of the whole axon is due to delta IIg being of the same order of magnitude as delta IIm. The degree of free swelling of axoplasmic gels was studied as a function of pH, salt concentration, and hydration radius of the anion of the salt used. The swelling increased with an increase in the reciprocal of the hydration radius, a decrease in salt concentration, and at pH below or above similar to 4.5. The nature of the constraints to the free swelling of axoplasm in axons immersed in seawater was studied. With the seawater employed, these constraints appeared to be due more to the retractive forces of the sheath than to delta IIm.


Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane. The role of phospholipids in the binding of [3H]tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A2 (Crotalus adamanteus and Naja naja) or phospholipase C (Bacillus cereus and Clostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A2, and with phospholipase C the lipid hydrolysis was about 60--70% for a 70--80% reduction in the binding activity. Phospholipase C from B. cereus and Cl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A2 but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A2. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.


A study on the toxicity of natural food dyes--toxicity and enzyme inhibition in Paramecium caudatum. The toxicity of 14 commercial natural dyes which are widely used as food additives in Japan was studied on Paramecium caudatum. Laccaic acid and capsanthin were found to be very toxic to Paramecium caudatum. Some of the commercially available carminic acid and crocin were also toxic. The inhibitory effect of natural food dyes on leucine aminopeptidase, acid phosphatase and esterase in vitro was proportional to the toxic effect of the dyes on the survival time of Paramecium caudatum. Analyses of the commercial natural food dyes by high performance liquid chromatography failed to identify the toxic components.


Dose-related response of mouse mamma to some phenothiazine drugs. The dose-related response of the mammary growth was studied in immature ICR-JCL female mice given 0.03 micrograms of estradiol per animal subcutaneously, and 0.005-1.0mg of either of the following phenothiazines (chlorpromazine, levometh iomeprazine, promethazine, perphenazine, thioridazine and propericiazine) per 10g of body weight was given orally once a day for 9 days starting on the 5th day after ovariectomy. The right second mamma of the chest was used to compare the hypertrophic affects. All the phenothiazines used produced mammary growth in the intensity sequence of thioridazine greater than chlorpromazine greater than promethazine greater than propericiazine greater than perphenazine greater than levomethiomeprazine. An optimal dose of each of the phenothiazines required to produce the maximum hypetrophy of the mammary gland was examined. From the results obtained, the relationship between the side-chain moiety of the compounds and the mammary growth activity could not be revealed.


Mandibular lesions in the Western Arctic caribou herd of Alaska. Lesions were noted in 7.0 and 4.4% of mandible pairs collected from the Western Arctic caribou herd of northwestern Alaska in 1959-61 and 1975-77, respectively. The prevalence of mandibular lesions in the 1959-61 collection is believed to be the highest reported in wild caribou herds of North America. The frequency of occurrence of mandibular lesions was highest in caribou 7 years of age and older, and there was a higher prevalence in adult males than in adult females. Trauma, dental abscesses, and periodontal disease were the probably cause of most lesions. Pathogenic bacteria were not isolated from mandibular lesions from an 11 year-old female. Thirty-three of 98 (33.7%) mandibles with lesions were missing one tooth, while ten (10.2%) were missing more than one tooth. The first molar (M1) was the most common tooth lost in association with lesions, although the loss of two or more teeth was more common among premolars than among molars.


Approaching long-term neuroleptic treatment of schizophrenia. Neuroleptic therapy can prevent some acute episodes and may improve the level of function in some cases of chronic schizophrenia. The hazards of tardive dyskinesia require a rigorous design for any long-term use of neuroleptics. The protocol includes narrow indications, demonstrations of efficacy and necessity, and arrangements for surveillance, cooperation, and emergency. Patient instruction improves the precision of medication use and may increase adaptive efforts during episodes.


Hormone secretion in alcohol-induced pseudo-Cushing's syndrome. Differential diagnosis with Cushing disease. We describe the clinical and biochemical characteristics of alcohol-induced pseudo-Cushing's syndrome in two patients with signs and symptoms of Cushing's syndrome and slight disturbances in liver function. An insufficient suppression of plasma cortisol to the overnight administration of 1 mg of dexamethasone was accompanied by an absent diurnal rhythm of plasma cortisol and an increased cortisol secretion rate. The plasma cortisol levels at 8 AM normalized during hospital admission (ie, alcohol withdrawal) at the same rate or parallel with the serum gamma-glutamyl transferase concentration. A normal increase of plasma cortisol, adrenocorticotropic hormone, and growth hormone in response to an insulin induced hypoglycemia was not compatible with the diagnosis Cushing's syndrome. However, the result of a differential diagnostic test with metyrapone was compatible with the presence of Cushing's disease. An erroneous diagnosis of Cushing's disease can be easily made in patients with alcohol-induced pseudo-Cushing's syndrome.


Feedback regulation of nephron filtration rate during pharmacologic interference with the renin-angiotensin and adrenergic systems in rats. Tubuloglomerular feedback has been defined as a mechanism in which changes in distal tubular sodium chloride delivery induce changes in glomerular arteriolar resistance. Experiments were performed in rats to test the hypothesis that the alterations in vasomotor activity are controlled by local hormonal mechanisms. Early proximal flow rate (EPFR), used as an index of filtration rate, was assessed at loop perfusion rates of 10 and 40 nl/min and during zero loop flow before and during intravenous administration of agents which interfere with the reninangiotensin or adrenergic systems. During infusion of the angiotensin (A) antagonists [Sar1,Ile8-]-AII or [Me2,Gly1,Ile8]-AII at doses ranging from 4.8 to 30.6 micrograms/kg . min, feedback response, expressed as percent change of EPFR during loop flow elevation from 3 to 40 nl/min, fell from a mean of 47.6 +/- 3.3% to 33.2 +/- 2.9% (P less than 0.05). Likewise, after administration of the converting enzyme inhibitor SQ 20881 in a dose ranging between 5.5 and 34.0 mg/kg, feedback response decreased from 48.5 +/- 2.1% to 25.9 +/- 1.9% (P less than 0.001) and returned to 43.1 +/- 5.1% after the inhibitory effect of SQ 20881 on the pressure response to angiotensin I had disappeared. Luminal application of [Sar1,Thr2]-AII (5mM) or of SQ 20881 (5 or 10 mM) had no effect on the feedback response. A significant reduction in the feedback response was noted also during intravenous infusion of propranolol (46.4 +/- 3.2% vs. 29.0 +/- 2.8%, P less than 0.001), whereas 6-OH-dopamine, reserpine, or phenoxybenzamine had no detectable effect. Our results are in agreement with the concept that the renin-angiotensin system may mediate feedback-induced resistance changes. In addition, circulating catecholamines may, in some unknown manner, act as modulators of the feedback response.


pH-Dependence of water and solute transport in toad urinary bladder. Stimulation of urea and water transport by vasopressin (ADH) appears to occur via independent pathways. We examined the effects of altering serosal or mucosal bath pH on transport of water, urea, and sodium. Compared to bladders with a serosal bath pH of 7.4 to 8.0, reducing the serosal bath pH to 6.8 led to a 60% fall in ADH-stimulated osmotic water flow, without decreasing the permeability of urea. Raising the serosal pH to 9.5 had the opposite effect: urea permeability was inhibited by 40% without altering water flow. Exogenous cyclic AMP-stimulated water and urea permeabilities were not dissociated, but were changed in the same direction by alterations in serosal pH: serosal acidification enhanced the effect of exogenous cyclic AMP on both urea and water, whereas the cyclic AMP effect on both was diminished by serosal alkalinization. This was especially marked for urea, suggesting that an alteration in the urea response to cyclic AMP may be particularly important in defining vasopressin-stimulated urea permeability as the serosal bath pH is altered. Mucosal acidification increased short circuit current but decreased both the urea and water response to ADH and 8-bromo-cyclic AMP. The response to cyclic AMP was less consistent. Mucosal alkalinization did not cause significant changes in either basal or stimulated transport. The data demonstrate distinct and separable effects of bath pH alterations on each of the transport systems examined.


Fractional extracellular space and fractional water content of various rat tissues at different extracellular pH values and in uremia. At different extracellular pH values fractional extracellular space (calculated from the distribution of 3H inulin) and fractional water content showed no significant differences. 72 h after nephrectomy both variables increased significantly. An exception to this was brain, where fractional extracellular space decreased and total intracellular water increased as a sign of brain oedema.


Comparison of regulated-breathing verses abbreviated desensitization on reported stuttering episodes. This study was an investigation of the regulated-breathing method for controlling stuttering as compared to a placebo-control method consisting of abbreviated desensitization training. The regulated-breathing procedure, given to 21 stutterers, taught the speaker to breathe smoothly and deeply, to pause at natural juncturing points, to plan ahead for the content of the speech, and to relax chest and neck muscles. Several general behavioral procedures were also used including relaxation training, self-correction for errors, social support, daily home practice, and response awareness, which are components of the general habit reversal procedure for diverse habits. Training was given in one or two sessions plus regular follow-up telephone calls. Daily self-recordings were obtained of the number of stuttering episodes during everyday speech, to determine the generalized effect of the treatment. The regulated-breathing method reduced the reported stuttering episodes by 94% on the first day after training and by 97% during the fourth week and the three-month follow-up. The control procedure reduced reported stuttering only slightly (about 10%). The results indicate substantial effectiveness of the regulated-breathing method for reducing reported stuttering episodes in everyday speech as compared with an alternative treatment of equal duration.


Tricyclic antidepressants and histamine H1 receptors. Tricyclic antidepressants and some structurally related compounds were tested for their ability to antagonize histamine H1 and muscarinic acetylcholine receptors of cultured mouse neuroblastoma cells. As a group, tertiary amine tricyclic antidepressants tended to be more potent than secondary amine drugs at both receptors. The most potent antihistamine, doxepin hydrocholoride, was about 4 times more potent than amitriptyline hydrochloride, about 800 times more potent than diphenhydramine hydrochloride, and about 8,000 times more potent than desipramine hydrochloride, the least potent tricyclic antidepressant at both the histamine H1 and the muscarinic acetylcholine receptors. All tricyclic drugs except desipramine hydrochloride were more potent as antihistamines than as anticholinergics. Doxepin hydrochloride and amitriptyline hydrochloride may be the most potent antihistamines known, and the antihistaminic potencies of these and the other tricyclic antidepressant drugs may relate directly to their ability to cause sedation and drowsiness in patients.


Genetical and cytological location of the structural parts coding for the first three steps of pyrimidine biosynthesis in Drosophila melanogaster. The rudimentary locus (r; X--55.3) of Drosophila melanogaster is shown to contain the structural sequences for the enzymes CPSase, ATCase and DHOase. The enzyme concentration in adult flies is correlated with the number of r+ copies in the genome. The expression of the locus follows the rules of the gene dosage compensation hypothesis when extracts of newly emerged males and females are compared.


Suppressor mutations causing partial reversion in the amiA region of Pneumococcus. Mutants of an aminopterin-resistant strain of pneumococcus possessing four different suppressor genes have been isolated after mutagenesis with 5-BUdR. The suppressed strains exhibit a partial revertant phenotype since the parental aminopterin resistance remained unchanged but the associated sensitivity to an excess concentration of the branched chain amino acids L-isoleucine, L-valine and L-leucine was diminished almost to the level of the wild-type strain C13. The suppressor mutations had therefore dissociated the two properties associated with a mutation in the amiA cistron, namely aminopterin resistance and isoleucine sensitivity. The suppressor genes reduced the sensitivity to isoleucine of a number of amiA mutants, but had no effect on the level of resistance to a number of unrelated genes conferring resistance to other antibacterial substances. The suppressor mutations themselves did not confer resistance to aminopterin. Mapping of the suppressor mutations by recombination analysis and by clonal analysis showed them to be intragenic lying in the region near to the amiA-r19, amiA-r23, amiA-r17 loci.


Trace element studies on four species of Fusarium. Trace element studies were carried out on four species of Fusarium: F. moniliforme, F. solani, F. poae and F. bulbigenum. Out of fifteen trace elements tested, Fe, Zn, Mn, Cu, Mo and B were found to be essential for growth and sporulation of all these species of Fusarium. Optimum concentrations in ppm of essential trace elements of these fungi were as follows: F. moniliforme Fe 1.0, Zn 0.1, Mn 0.1, Cu 0.1, Mo 0.1, B 1.0-10.0; F. solani Fe 1.0, Zn 10.0, Mn 0.1, Cu 0.01, Mo 1.0, B 0.1; F. poae Fe 1.0, Zn 0.1, Mn 0.1, Cu 1.0, Mo 0.1, B0.1, F. bulbigneum Fe 10.0, Zn 1.0, Mn 1.0, Cu 1.0, Mo 0.01, B 1.0. Concentrations higher than the optimum were inhibitory to the respective fungi.


[Formation of higher alcohols by Saccharomyces carlsbergensis from branched-chain amino acids and their keto analogs]. A new method of chemical synthesis of alpha-ketoisocaproic acid (the keto analogue of L-leucine) is described. It has been shown that the resting cells of Saccharomyces carlsbergensis 776, in the stationary state of biomass, produce mainly higher alcohols: isobutanol from L-valine and its keto analogue; optically active amylol only from L-isoleucine and its keto analogue; isoamylol only from L-leucine and its keto analogue. "Nonspecific" formation of n-propanol from L-valine, L-isoleucine and their keto analogues, as well as that of isobutanol from L-isoleucine and its keto analogue, has been also found at pH 7.0. Formation of higher alcohols from alpha-keto acids has an acidic pH optimum while that from L-amino acids has a neutral or a weakly alkaline pH optimum. Formation of isobutanol from L-valine is an exception. The dependence of higher alcohol formation on the pH and the kinetics of their accumulation suggest that higher alcohols are produced from L-amino acids in at least three sequential reactions: transamination, decarboxylation of the keto analogue being formed, and reduction of the aldehyde; formation of higher alcohols from alpha-keto acids involves two reactions: decarboxylation and reduction. Transamination and decarboxylation are limiting steps in the process in the former case, and decarboxylation in the latter.


[Lactate oxidation by Hansenula anomala cells]. Lactate oxidation by freeze-dried cells of Hansenula anomala was studied in the presence of exogenous electron acceptors, potassium ferricyanide (PF) and phenazine metosulfate (PMS). The product of oxidation is pyruvate. The apparent Michaelis constants for lactate, PF and PMS are 1.2, 0.6 and 0.05, respectively. The pH optimum of lactate oxidation is 8.0. The freeze-dried cells of the yeast are inactivated by 5 mM bromopyruvate by 50% within 1.5 min. Treatment of the intact cells with ultrasound activates them. The respiratory function of the cells is characterized with a KM(app.) of 0.05 mM in the presence of lactate. PF has no effect on respiration. A scheme is proposed for lactate oxidation by the cells with damaged membranes whose amount after lyophilization is 10--20%.


[Cell adhesion in a chemostat culture of Candida utilis under the influence of supraoptimal temperature and elevated acidity]. The effect of the supraoptimal temperature (38, 40 degrees C) on the chemostat culture of Candida utilis was studied. The above factor caused a part of the biomass to float as a thin layer of foam to the surface of the medium. After an hour, the concentration of the cells on the surface could be four times as high as that within the medium. The content of protein was the same in the cells taken from the surface and from the depth. Singular cells or their groups (2 or 4--8 cells) were found deep in the medium whereas cells on the surface were aggregated forming conglomerates of 20--100 and more cells. The simultaneous action of the elevated tmperature and the acid pH value made flotation of cells onto the surface more stable and protracted (it could be maintained in a chemostat for weeks).


[The effect of a beta-adrenergic blocking agent on driving capability (author's transl)]. The effect of Betadrenol, a beta-adrenergic blocking agent, was studied by means of traffic psychology tests in 29 male patients with cardiovascular disorders. The age range was 29 to 60 years. After a 7-day wash-out period, each subject received either 3 x 100 mg Betadrenol or a placebo daily during three days. Subsequently every patient was given 3 x 100 mg of Betadrenol daily during another ten days. The short-term double-blind study revealed no impairment of driving performance. The results of the open study showed significant improvements, especially in concentration and speed of reaction.


[Candida mycoderma growth inhibition with phenol and the autoselection of resistent forms under continuous pH-stat cultivation]. The effect of phenol on the growth rate and respiration was studied with the yeast Candida mycoderma cultivated in the pH-static conditions with continuous recording of the principal kinetic parameters of the population. The kinetics of growth inhibition with phenol was studied. Adaptation of the culture in terms of the growth rate and the rate of oxygen uptake was detected within 10--15 hours of cultivation. A new strain of C. mycoderma Phen. R. isolated using the technique of autoselection upon continuous cultivation for a long period of time, at a phenol concentration of 1.5 g/l in the growth medium, had an elevated growth rate (2.2 times higher) in these conditions as compared with the parent culture and required less oxygen.