The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.


[Protective effect of thymidine in relation to the cytostatic action of cyclic adenosine-3', 5'-monophosphate in mammalian cell cultures].

Under the action of lmM of cyclic adenosine-3',5'-monophosphate (cAMP) the survival and the rate of reproduction of the cells of Chinese hamster in the culture decreased in comparison with control to 27 and 42%, respectively. Addition of 0.02 mM thymidine together with cAMP almost completely eliminated the cytostatic action of the latter. Thymidine also prevented the cytostatic effect of noncyclic 5'-AMR, but failed to influence the death of the cells with the action of dibutyril cAMP and theophylline. Thymidine failed to prevent the inhibitory action of cAMP on the mutant strains of mouse cells defective by thymidinekinase. A conclusion was drawn that in the concentrations under study thecytostatic action of the exogenous cAMP on the cells of mammals served as the sequence of splitting to 5'-AMP in the culture medium and was realized by blocking one of the stages of TMP synthesis.


Echocardiographic pattern of right atrial tumour motion.

Propagation of Wilm's tumour to the right atrium was diagnosed by echocardiography. The tumour prolapsed into the right ventricle during each atrial systole. The presence of the tumour and its motion were confirmed by cardiac catheterization, cineangiography, and surgical exploration. Successful resection of the tumour was accomplished. Echocardiography was helpful in detecting the presence of the right atrial tumour and accurately reflected the pattern of its atrioventricular motion.


Comparison of tumour susceptibility among various organs of foetal, young and adult ICR/Jcl mice.

Urethane was found to be uniformly distributed in all the major organs of foetal, young and adult ICR/Jcl mice and then to disappear rapidly as measured by the incorporation of urethane-carbonyl-14C, thus permitting the accurate comparison of tumour susceptibility of cells in various organs of mice at different ages. Lung tumour frequency (tumours/lung) was significantly higher in mice treated with urethane when young (21 days old) and adult (63 days old) than in those treated in utero (Days 11-19 of gestation). When relative sensitivity of a lung cell was calculated as the ratio of average number of tumours per lung per mg of lung at the time of treatment, however, a lung cell of the foetus was more sensitive to urethane than that of the young and adult. Hepatomata were induced significantly only when male foetuses and neonates were exposed to urethane. The offspring exposed to urethane on Days 11-16, however, developed hepatomata in lower incidence than those exposed on Days 14-19, whereas the previous investigation by the author revealed that Days 11-13 correspond to the stage most sensitive to hepatocarcinogenesis. This contradiction was due to the occurrence of testicular hypogenesis (chemical castration) in all offspring of the former group. Differentiating female gonad and rapidly proliferating blood vessels of the placenta and deciduum were also sensitive to tumour induction by urethane. Thus, high tumour susceptibility of rapidly proliferating and undifferentiated cells suggests that some initiating events in the process of carcinogenesis may occur during or after DNA replication. Leukaemia induction in the young mice, but not in the foetus, remains to be elucidated.


Accidental exposure to isocyanate fumes in a group of firemen.

A total of 35 firemen involved in fighting a fire in a factory in which polyurethane foam was made were exposed to fumes of toluene di-isocyanate from two large storage tanks which were damaged during the fire, resulting in massive spillage. Most of the men experienced symptoms during the fire or during the three weeks after it. The symptoms were mainly gastrointestinal, respiratory, or neurological. Altogether 15 men described gastrointestinal symptoms which subsided within two days of onset. Respiratory symptoms were described by 31 men and were most pronounced during the three days after the fire, thereafter tending to improve. The neurological findings are described separately. When the men were reviewed at six months there was a suggestion that some of them might have sustained long-term damage to the respiratory tract, and almost four years later 20 men had persistent respiratory symptoms. Serial measurements of ventilatory capacity revealed a marked decline in the first six months although this was not sustained.


[New concepts on the etiopathogenesis of diarrhea].

The author discusses new knowledge concerning E. coli enterotoxins and Duovirus in the production of diarrheal disease.


Neurological complications after a single severe exposure to toluene di-isocyanate.

A total of 23 men complained of neurological symptoms after a single severe exposure to toluene di-isocyanate. Effects of exposure were immediate in five men and consisted of euphoria, ataxia, and loss of consciousness. These men and nine others complained of headache, difficulty in concentration, poor memory, and confusion during the next three weeks. Four years later it was found that nine further men had experienced symptoms that they had not been aware of at three weeks. In all, 13 men still complained of poor memory, personality change, irritability, or depression after four years. Psychometric testing showed a selective defect for relatively long-term recall in those with persistent symptoms at four years.


Cooperativity and noncooperativity in the binding of NAD analogues to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Using NAD analogues as ligands, the structural requirements for negative cooperativity in binding to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase were examined. Although the affinity of nicotinamide hypoxanthine dinucleotide is considerably lower than that of NAD+, it also binds to the enzyme with negative cooperatively. Two pairs of nicotinamide hypoxanthine dinucleotide binding sitess were distinguished, one pair having an affinity for the analogue which is 15 times that of the second pair. Negative cooperativity is also found in the Km values for the analogue. Thus modification of the adenine ring of NAD+ to hypoxanthine does not abolish negative cooperativity in coenzyme binding. Adenosine diphosphoribose binding to the same enzyme shows neither positive nor negative cooperativity, indicating that cooperativity apparently requires an intact nicotinamide ring in the coenzyme structure, under the conditions of these experiments. Occupancy of the nicotinamide subsite of the coenzyme binding site is not necessary for half-of-sites reactivity of alkylating or acylating compounds (Levitzki, A. (1974), J. Mol, Biol. 90, 451-458). However, it can be important in the negative cooperativity in ligand binding, as illustrated by adenosine diphosphoribose which fails to exhibit negative cooperativity. Occupancy of the adenine subsite by adenine is important for stabilization of the enzyme against thermal denaturation. Whether the stabilization is due to an altered conformation of the subunits or stabilization of the preexisting structure of the apoenzyme cannot be determined from these studies. However, nicotinamide hypoxanthine dinucleotide does not contribute to enzyme stability although it serves as a substrate and shows negative cooperativity.


Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid.

Six enzymes involved in the conversion of aspartate to threonine have been extracted from Escherichia coli and separated from each other. Two of these enzymes, aspartokinase and homoserine dehydrogenase, have also been partially purified from Rhodopseudomonas spheroides. In an attempt to determine whether small changes in the kinetic properties of individual enzymes are important to the regulation of metabolic flux through a coupled reaction system, the partially purified enzymes were recombined in a variety of ways under reaction conditions designed to resemble the in vivo situation. These conditions include: use of an entire metabolic system rather than a single reaction; high enzyme concentrations at the same relative concentrations as found in the cell; and low, steady-state concentrations of substrates and products. Metabolic flux was followed spectrophotometrically and the concentrations of aspartic semialdehyde, hemoserine, O-phosphohomoserine, and threonine were measured. The results indicate that the threonine concentration is of major importance in regulating metabolic flux by inhibiting aspartokinase, the first reaction in threonine in the pathway. When threonine-insensitive aspartokinases were used, concentrations reached higher levels and the rate of NADPH oxidation remained higher. The fact that neither aspartic semialdehyde nor homoserine accumulated as the threonine concentration increased and the lack of correlation between changes in metabolic flux and ADP/ATP or NADPH/NADP ratios indicate that more subtle forms of metabolic regulation, such as "reverse cascade", secondary feedback sites, or "energy charge", are of little regulatory importance in this isolated, metabolic system. The results also emphasize the need for caution in projecting in vivo control mechanisms from in vitro experiments.


In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.


Association of methanol and ethanol with heme proteins.

The behavior of ferrihemoglobin and ferrimyoglobin in widely varying concentrations of the lowest four alcohols has been studied by optical and electron paramagnetic resonance absorption spectroscopy. Methanol and ethanol, at concentrations too low to cause general conformational destabilization of the protein, produce both optical and electron paramagnetic resonance absorption spectral changes in ferrihemoglobin. These changes arise from equilibrium associations, characterized by dissociation constants at 25 degrees C of about 40 and 200 mM, respectively, for the methanol-ferrihemoglobin and ethanol-ferrihemoglobin complexes so formed. Other optical spectral changes appear when the methanol concentration exceeds 3.5 M and the ethanol, 1.0 M. At concentrations lower than 0.5 M, 1- and 2-propanol produce spectral changes of this second kind. At room temperature no optical evidence has been found that the propanols associate with ferrihemoglobin in the manner of methanol and ethanol. Methanol and ethanol at low concentration have specific effects, characterized by electron paramagnetic resonance spectral differences, upon ferric alphaSH chains. All four alcohols, over a wide range of concentrations, reduce the symmetry of electron paramagnetic resonance spectra from frozen solutions of ferrihemoglobin; even at the high end of this concentration range, none  of the alcohols reduces the symmetry of electron paramagnetic resonance spectra from frozen ferrimyoglobin. Ferrimyoglobin and catalase association with methanol is measurable optically; the binding is about five and sixty times weaker, respectively, for these two proteins as compared with ferrihemoglobin.


Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.


Sequence composition of the template-active fraction of rat liver chromatin.

Rat liver chromatin has been separated into nuclease-sensitive and -resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg2+ -soluble and -insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min at 10 enzyme units/A260 unit of chromatin at pH 6.6), 11% of the input chromatin DNA is found in the Mg2+ -soluble fraction. This DNA has a weight-average single-strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of nonrepetitive DNA sequences and a subset of families of middle repetitive sequences. This demonstrates the nonrandom distribution of repetitive and single copy sequences in the Mg2+ -soluble fraction of chromatin. Previous studies have shown that the Mg2+ -soluble fraction is enriched in nonrepeated  sequences which are transcribed in vivo (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). We now report that the Mg2+ -soluble fraction of liver chromatin contains a low proportion of sequences in common with the Mg2+ -soluble fraction of brain chromatin. Thus, fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained.


Divergent effects of adrenocorticotropin and melanotropin on isolated rat and rabbit adipocytes.

The stimulation of lipolysis in isolated rat and rabbit fat cells by adrenocorticotropin (ACTH) and alpha-melanotropin has been studied. The concentration of alpha-melanotropin required for half maximal stimulation is 0.23 times that of ACTH in rabbit adipocytes but as high as 1140 times that of ACTH in rat fat cells. Chemical modification of the tryptophan residue in ACTH and melanotropin resulted in a loss of lipolytic activity in rat adipocytes and an increase in lipolytic potency in rabbit fat cells. These differences between rat and rabbit fat cells were evident when stimulation of cyclic AMP synthesis was measured in isolated cells or ghosts. The results are discussed in terms of the difference in the hormone receptors of the fat cells of the two species.


Comparison of the steroidogenic and melanotropic activities of corticotropin, alpha-melanotropin and analogs with their lipolytic activities in rat and rabbit adipocytes.

The ability of alpha-melanotropin and a series of synthetic peptides related to adrenocorticotropin (ACTH) to stimulate steroidogenesis in isolated rat adrenal cells, melanin dispersion in frog melanophores and lipolysis in rat and rabbit fat cells have been studied. It was found that the steroidogenic activity closely paralleled the lipolytic activity of these peptides in rat fat cells, whereas the melanocyte stimulating activity paralleled the lipolytic activity in rabbit fat cells. These results indicate that the structural requirements for stimulating steroidogenesis in isolated rat adrenal cells and lipolysis in isolated rat fat cells are quite similar. The structural features required for eliciting lipolysis in rabbit fat cells appear to be very similar to those necessary for stimulating frog melanophores. The possibility that regulation of lipid metabolism in the rabbit may be a new function acquired by melanotropin is discussed.


Roles of alpha and beta adrenergic receptors in control of glucose oxidation in hamster epididymal adipocytes.

Oxidation of [14C] glucose in isolated epididymal adipocytes from Golden hamsters was stimulated by isoproterenol, epinephrine and norepinephrine, which all interact with beta-adrenergic receptors and by adrenocorticotrophic hormone. In contrast alpha-receptor agonists, such as phenylephrine, methoxamine or clonidine did not increase basal glucose oxidation. The beta-adrenergic blocking drug propranolol inhibited both lipolysis and glucose oxidation when these had been stimulated by isoproterenol, epinephrine or norepinephrine. Conversely, the alpha-adrenergic blocking drugs phentolamine and phenoxybenzamine did not influence lipolysis or glucose oxidation when isoproterenol provided the stimulus and increased both lipolysis and glucose metabolism in the present of either epinephrine or norepinephrine. All alpha-adrenergic agonists tested (phenylephrine, methoxamine and clonidine) lowered lipolysis and glucose oxidation isolated adipocytes exposed to isoproterenol. However, when adrenocorticotropin provided the stimulus for glucose oxidation and lipolysis, only clonidine produced a significant reduction in lipolysis and glucose oxidation. None of the alpha-agonists influenced glucose metabolism which had been increased by insulin. These data confirm the presence of both alpha and beta adrenergic receptors on hamster epididymal adipocytes and suggest that they exert antagonistic influences on lipolysis and glucose oxidation. These data are also consistent with the view that adrenergic stimulation of glucose oxidation and lipolysis in adipocytes are both mediated through beta receptors.


Maternal-perinatal interrelationships of vitamin D metabolism in rats.

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.


The dependence of specific nuclear binding of glucocorticoids by rat thymus cells on cellular ATP levels.

The specific, saturable, nuclear binding of corticosteroids by rat thymus cells has been studied. Both the accumulation and the retention of corticosteroids by the nuclei of rat thymus cells at 37 degrees C were dependent upon the metabolic state of the cells. Treatment of cells with 2,4-dinitrophenol in the absence of glucose inhibited both accumulation and retention  in the cell nucleus; these effects could be only partially prevented by the addition of glucose. The presence or absence of glucose also influenced the level of nuclear binding in the absence of 2,4-dinitrophenol, and in all cases the levels of nuclear binding of corticosteroids were correlated with cellular ATP levels. The effects of other reagents on levels of ATP and cyclic AMP also showed that significant changes in the level of nuclear-bound steroid were always accompanied by parallel variations in ATP levels, although there were no such correlations with levels of cyclic AMP. The partial restoration of nuclear binding by glucose in cells previously treated with 2,4-dinitrophenol was unaffected by cycloheximiede although protein synthesis was inhibited. Possible mechanisms by which the corticosteroid receptor is converted to a form capable of binding steroids by a reaction which requires energy but not de nova protein synthesis are discussed.


Optimum conditions for the binding of insulin to its receptor.

The specific binding of insulin to the membranes from lactating mouse mammary gland was studied as a model of hormonereceptor type of binding. The basic ingredients of binding, the concentration of receptor protein and the concentration of labeled insulin were mainly studied. The characteristic changes in specific binding were followed, the adequate regression equation was draen and the optimum conditions of binding were established for further experiments. The expediency of applying shortened orthogonal plans, regression and analysis and graphic conture analysis were proved.


The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures.

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal calf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous addition of 10(-2) M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and is noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.


The absence of 2,3-diphosphoglycerate from myocytes, hepatocytes and adipocytes.

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also presented which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.


Secretory processes, carbohydrate and lipid metabolism in isolated mouse hepatocytes. Aspects of regulation by glucagon and insulin.

The procedure of Berry and Friend for isolation of intact hepatocytes has been adapted to mouse livers. The ultrastructure of these cells was satisfactorily preserved. Isolated mouse hepatocytes secreted proteins and triacylglycerols. These secretory processes were inhibited by colchicine, indicating a likely involvement of the microtubular system for their normal occurrence. Ultracentrifugation of medium incubated with hepatocytes, followed by electrophoresis and electron microscopic examination of the floating fraction (density less than 1.006) allowed to conclude that secreted triacylglycerols were very low density lipoproteins. Glycogenolysis and lipogenesis were stimulated or inhibited, respectively, by low concentrations of glucagon (10(-10) M). Other metabolic parameters were influenced by the hormone but were less sensitive to its action. Inhibition of lipogenesis by glucagon was associated with a decrease in acetyl CoA carboxylase activity. This decrease does not appear to be related to intracellular fatty acyl-CoA accumulation secondary to hepatic lipase activation by the hormone. Insulin was effective alone or counteracted glucagon effects on lipogenesis or glycogenolysis only when exposure of cells to collagenase was held minimal. This suggests that, during isolation of hepatocytes, insulin receptors may, for unknown reasons, be more fragile than those of glucagon.


Activation of protein kinase(s) by glucagon and cyclic AMP in the rat liver. Relationship to metabolic effects.

Although it is known that protein kinases are activated by cyclic AMP, the role of the activated kinase in the gluconeogenic response to cyclic AMP is not known. Therefore, we examined whether the inhibition of the gluconeogenic response in the liver is due to an interference with the activation of protein kinase in the following situations: (1) adrenalectomy, (2) Na+-free perfusate, (3) administration of local anesthetic. We measured protein kinase activity indirectly by measuring incorporation of 32P into proteins of the perfused liver, and directly by measuring the enzyme activity. We found no significant inhibition of activation of protein kinase in the above experimental conditions. It seems that in the intact liver, activation of protein kinase by itself is not sufficient to evoke metabolic responses. In order to clarify whether the requirement for ion redistribution is specific for the gluconeogenic response or not, the lipolytic and antilipogenic effects of glucagon and cyclic AMP were examined. Na+-free perfusate, local anesthetic or high K+ did interfere with the lipolytic and antilipogenic responses to these agents just as it interfered with the gluconeogenic response. It is likely that ion redistribution evoked by glucagon and cyclic AMP is essential to the expression of most, if not all, metabolic effects.


Demonstration of adenylate cyclase activity in human red blood cell ghosts.

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine 3',5'-monophosphate (cyclic AMP) - 2 h-1 - mg-1 protein. This enzymatic activity is strongly stimulated by NaF and 5'-guanylimidodiphosphate, is slightly stimulated by epinephrine, norepinephrine, isoproterenol, and prostaglandin E1 and is inhibited by calcium. The hormone stimulation is not potentiated by 5'-guanylylimidodiphosphate.


Premature induction of amylase in pancreas and parotid gland of growing rats by dexamethasone.

Studies were made on changes in the contents of alpha-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal development, on the premature induction of this enzyme by hormones and on the existence of specific glucocorticoid receptors in these tissues. The amylase content in the pancreas increased from the 9th day after birth and reached the adult level on the 28th day, its content in the parotid gland increased rapidly from the 16th to the 28th day after birth and then rose more gradually to the adult level. Injection of dexamethasone into rats 6--8 days after birth induced increase in the amylase of the pancreas but not the parotid gland. However, injection of dexamethasone into weanling rats 21--23 days after birth resulted in precocious induction of amylase in both tissues. Specific glucocorticoid receptors were detectable in the parotid gland of rats from 6 days after birth but were almost undetectable in the pancreas until adolescence.


Acute effects of oral and intravenous ethanol on rat hepatic enzyme activities.

1. Oral administration of ethanol (3 ml) of 95% in 12 ml total volume over a two day period) significantly decrease plasma glucose and insulin levels and the activities of two key gluconeogenic enzymes, pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) and fructose diphosphatase, (D-Fru-1,6-P2 1-phosphohydrolase, EC 3.1.3.11), and one glycolytic enzyme, fructose-1,6-P2 aldolase (Fru-1,6-P2 D-glyceraldehyde-3-P lyase, EC 4.1.2.13). In each instance, the administration of 2400 mug daily of oral folate in conjuction with the ethanol prevented these alterations in carbohydrate metabolism. 2. Intravenous injection of ethanol produced a rapid decrease (within 10--15 min) in the activities of hepatic phosphofructokinase, (ATP:D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase, (ATP:pyruvate phosphotransferase, EC 2.7.1.40), fructose diphosphatase and fructose-1,6-P2 aldolase. 3. Intravenous ethanol significantly increased hepatic cyclic AMP concentration approximately 60% within 10 min, while oral ethanol did not alter hepatic cyclic AMP concentrations. 4. These data confirm the known antagonism ethanol and folate and suggest that oral folate might offer a protective effect against hypoglycemia in rats receiving ethanol.


Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine.

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition casued by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzyme synthesis as well as inhibition of activity.


Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanediol metabolism.

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for the enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.


Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system.

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 X g supernatant was prepared and the receptor and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at -5 degree C. The chromatin accumulated part of the complex at incubations at 4 degrees C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4 degrees C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07--0.25 nM).


Phospholipases and the effect of prolactin on uridine incorporation into RNA in mammary gland explants of mice.

The possible effects of phospholipase A and phospholipase C on the rate of uridine incorporation into RNA in mammary gland explants of mice were tested. Phospholipase C had no effect on the rate of uridine incorporation, but it did suppress the action of prolactin on this metabolic parameter. In contrast, phospholipase A was found to stimulate the rate of uridine incorporation into RNA in a manner similar to that of prolactin. The time-courses for the onset of the prolactin and phospholipase A effects were the same. Also, the phospholipase A effect was nonadditive to the effect produced by a maximally stimulatory concentration of prolactin. Finally it was observed that, like the prolactin effect, the phospholipase A effect was abolished by incubation with dibutyryl cyclic AMP, theophylline, quinine, indomethacin and prostaglandin E1. Further, the phospholipase A effect was nonadditive to the prolactin-like effects produced by the cyclic GMP, prostaglandin F2alpha or arachidonic acid. These data therefore suggest that prolactin and phospholipase A stimulate RNA synthesis in mammary gland explants via similar processes.


The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system.

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed. The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. EO2/O2=-0.33 V and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of O2/O2 is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O2 in biological processes of O2 fixation.


Compositional characteristics of a chloroform/methanol soluble protein fraction from spinach chloroplast membranes.

Extraction of an aqueous suspension of spinach chloroplast lamellae with a chloroform/methanol mixture leads to solubilization of about 1/3 of the total membrane protein. Amino acid analysis of the chloroform/methanol-soluble protein shows that this fraction is largely enriched in the hydrophobic residues proline, leucine, alanine and phenylalanine and considerably depleted in polar amino acids, namely lysine and arginine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material reveals the presence of a variety of low molecular weight polypeptides (molecular weight less than or equal to 25 000), with more than 50% of the total fraction being contributed by a 25 000 dalton band. This band, which accounts for about 25% of the total chloroplast lamellar protein, has recently been identified as the main component of the light-harvesting chlorophyll-protein complex. The physiological role of most of the chloroform/methanol-soluble protein fraction is not known at present. From its chemical properties and apparent biological inertness, we propose that it plays mainly a structural role in situ, interacting with the lipid moiety of the chloroplast membrane. The material insoluble in the aqueous chloroform/methanol mixture is largely enriched in manganese, iron, cytochrome and water-soluble proteins, such as chloroplast coupling factor and ribulose diphosphate carboxylase.


PRoperties of the low-temperature photosystem I primary reaction in the P-700-chlorophyll alpha-protein.

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.


The mobile receptor hypothesis and "cooperativity" of hormone binding. Application to insulin.

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone. A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from "negative cooperativity." (2) Receptor occupancy and biological response may be coupled in a non-linear fashion. By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of "cooperativity." These considerations may also be pertinent to interpretations of other hormone-receptor systems and of various ligand-macromolecule interactions.


The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase.

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient. The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified bisolateral plasma membranes was 13-fold. F--activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5'-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.


The effect of short chain fatty acids on transmural electrical potential across rat small intestine in vivo.

Short chain fatty acids suddenly produce a phasic increase in transmural electrical potential difference (PD) when placed in the lumen of rat small intestine in vivo. With concentrations of propionate ranging from 50 muM to 1000 muM the amplitude of the response in jejunum is about 5.5 mV. The concentration giving half this effect is about 20 muM. With 10 mM propionate the duration of the response is 3-5 min; after this, PD again equals the control value and the gut is refractory to further additions. Removing propionate from the mucosal surface produces no change in PD, but does restore responsiveness to subsequent exposure to short chain fatty acids. This effect is independent of a variety of other alterations in PD such as those caused by sugars, amino acids, bile salts, theophylline, prostaglandins, and ATP. Mechanism and significance of this surprisingly sensitive response remain obscure.


Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study.

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label (3'-spiro(2'=(N-oxyl-4',4'-dimethyl-oxazolidine))5alpha-cholestane) and a fatty acid spin label (4',4'-dimethyloxazolidine-N-oxyl derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55 degrees C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron miccroscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.


Hormone action at the membrane level. V. Binding of (+/-)-[3H]isoproterenol to intact chicken erythrocytes and erythrocyte ghosts.

The binding of (+/-)-[7-3H]isoproterenol to intact chicken erythrocytes has been investigated by a rapid centrifugation technique. The binding is displaceable by a one thousand-fold excess of cold isoproterenol and consists of two fractions, only one of which is inhibitable by the beta antagonist (--)-propranolol. The total displaceable binding to intact cells amounts to 80 or 127 molecules per cell at a (+/-)-isoproterenol concentration of 0.4 muM depending on the method employed to analyze the binding. Under similar conditions, the total displaceable binding to isolated membrane ghosts is 12600 molecules per cell. The propranolol-inhibitable binding to intact cell reaches saturation within 5 min at 4 degrees C and gives by scatchard analysis a maximum binding of 108 molecules per cell and with a KD of 0.4 muM. 50% inhibition of binding is obtained with 0.3 muM unlabeled (--)-isoproterenol as compared to 20 muM unlabeled (+)-isoproterenol. The binding of isoproterenol thus shows a marked stereospecific preference for the (--)-isomer.


Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.


Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.


Localization of enzymes in specialized regions of the microsomal membrane.

Microsomal vesicles were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patches in the microsomal membrane.


The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.


The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.


[Study, by an enzymatic method, of the isomers of L-proline (3H-4) and of the effects of preservation].

By use of procollagen proline hydroxylas which catalyses the substitution of the hydrogen in the trans position on carbon 4 with an hydroxyl on L-proline residues of procollagen, we have shown that the L-[3H-4] proline product of C.E.N. Saclay contained 29 per cent of the trans and 71 per cent of the cis form. It was also possible to verify the molecular stability of the isomers and that a small amount of tritium was randomly linked to the whole molecule.


[The action of calcium on the step of collagen metabolism, catalyzed by procollagen-proline hydroxylase].

Calcium effects on procollagen-proline-hydroxylase activity of chick embryo tibias was studied. Serial preincubation of the natural substrate, the enzymatic preparation or the mixture of both with and without calcium showed that calcium decreased procollagen-prolin-hydroxylase affinity for procollagen, decreased the maximum velocity of the reaction by binding to the substrate and by increasing the thermosensitivity of the enzyme -- substrate complex. Calcium could also increase the maximum velocity by dissociation of an enzyme -- inhibitor ligand complex. The resulting decrease of the overall reaction rate has been discussed in the context of the relationship between collagen metabolism and calcium fixation during calcification.


[Formation of adducts of pyridine nucleotides in the active center of swine lactate dehydrogenase (isoenzyme M4)].

Isozyme M4 of pig lactate dehydrogenase (LDH-M4) catalyzes reaction of NAD-adduct formation with a nucleophylic agent that is perhaps OH--ion. The T 1/2 of the reaction is 10-30 sec at concentration NAD 2,0-10(-3) M, LDH-M4 50 gamma/ml at pH greater than 8. Initial velocity and limit of the reaction increase at high LDH-M4, NAD and OH--ion concentrations. Pyridine-3-aldehyde and 3-acetyl pyridine analogs of NAD forms fluorescent adducts too, but at OH--ion concentration approximately 0,01 of that in the case of NAD reaction. Isoelectrical point of LDH-M4 determined by isoelectrofocusing method is 8,65 +/- 0,04 pH unit.


[Pyridoxal kinase activity and pyridoxal-P concentration in mammalian tissues under normal and experimental conditions].

The activity and the distribution of pyridoxal kinase in rat and mouse tissues are studied. The data obtained testify the presence of a relative excess of pyridoxal kinase in all the organs studied, which probably causes a high rate of pyridoxalphosphate (PLP) biosynthesis under comparatively low vitamin B6 concentration. A correlation between the level of pyridoxal kinase activity and the content of PLP in rat brain and liver during ontogenesis is observed. The activity of pyridoxal kinase and the content of PLP are shown to be sharply increased in liver of rats received a protein-rich diet. Bilateral adrenalectomy resulted in the decrease of absolute and specific enzyme activities in rat liver by 20--30%. The content of PLP in mouse brain and liver was sharply decreased under experimental B6-avitaminosis while the content of pyridoxal kinase practically did not change. The injection of vitamin B6 rapidly normalized the PLP content in mouse tissues. The data obtained show that under physiological conditions the functional activity of pyridoxal kinase may be regulated in tissues by enzyme and substate contents. Some aspects of vitamin B6 metabolism in mammals are considered. It is concluded that in body the pyridoxal catabolism connected with its phosphorylation by pyridoxal kinase and the formation of pyridoxalphosphate.


[Adrenal cortex cytochrome c].

The method of preparation of highly purified cytochrome c from bovine adrenal cortex is described. Absolute spectra of the protein in reduced and oxidized states and some its physico-chemical properties are investigated.


[Distribution and activity of pyridoxal kinase in human brain during ontogenesis].

The activity of pyridoxal kinase was sharply increased in whole brain tissue of human, embryos and fetuses within 6-11 weeks of development. In brain stem the maximal values of the enzyme activity was observed at early stages of prenatal development of fetuses. The activity of pyridoxal kinase was increased in cerebral cortex and in the limbic system up to complete maturation of fetuses. It correlated with the fetus age within 14-40 weeks of development as calculated per 1 g of tissue wight or 1 mg of protein. The enzyme is distributed evenly in brain of newborns, babies and adult people. Its activity in grey cortex substance is higher, than in white one. There are 2-10-fold individual fluctuations of pyridoxal kinase activity in brain of people without CNS pathology. In newborns, having prolonged hypoxy at prenatal period, the enzyme activity was on the average by 70-80% lower at different brain parts than in newborns which had no primary asphyxia. A low pyridoxal kinase activity (not more than 1-5% as compared with its normal level) was observed in different brain parts of a child affected by focal gliosis and epilepsy.


[The interaction of pyridine-adenine-dinucleotides containing a hydrazide group with swine muscle lactate dehydrogenase].

Hydrazide group of 4-substituted NAD analogues is shown to interact with functional groups of substrate-binding site in double complexes with pig muscle lactate dehydrogenase (isoenzyme M4). The lactic acid residue, which is structurally incorporated into NAD analogue, improves slightly the binding of dinucleotide, while 2,2,6,6-tetramethylpiperidine-1-oxyl residue considerably decreases the firmless of binding. The comparison of the inhibitory ability of oxamate, incotinic acid hydraxide and their spin-labelled derivatives indicates the restricted and stiff sizes of a substrate-binding site.


[Kinetics and regulatory properties of the disulfide reductase enzyme from mouse liver].

Disulfide reductase (DSR) of mice liver supernatant is kinetically demonstrated as associating-dissociating oligomeric protein with positive homotropic cooperativity for the substrate. Cyclic 3',5'-AMP (10(-11)--10(-5) M) activates DSR and increases V, but does not change either [S]0,5, nor nH and does not shift the plot of specific activity versus the enzyme concentration. ATP, GTP, UTP, CTP, protamine, histone, Mg2+, Ca2+, EDTA (but not adenosine, 5'-AMP, 2'3'-AMP, ADP beef serum albumin) activated DSR. The effects of different modifiers are not summed up. Preincubation is essential for the action of the majority of the activators. Heating for 8 minutes at 55 degrees C desensitized completely DSR to all the modifiers without changing its catalytic activity, [S]0,5 and nH values. Possible mechanisms of activation of DSR, especially the involvement of protein kinase, are discussed.


[The stimulating effect of cyclic AMP, glucagon and insulin on guanidine acetate-N-methyltransferase activity in rat liver and pancreas].

N6,O2'-dibutyrylcyclo-3',5'-AMP injected to intact rats alone or in combination with theophylline increases the activity of guanidine acetate methyltransferase (GAMT) in liver and pancreas. Cyclic 3',5'-AMP and its dibutyryl analog administered immediately or two hours after the suturing of common bile duct (SCBD) stimulate the increase of pancreatic GAMT activity 2-3 fold. Glucagon, injected intraabdominally simultaneously with SCBD and administration of theophylline, dramatically increases the theophylline effect on the GAMT activity. The freezing of rat pancreas pretreated witn secretin, a hormone structurally similar to glucagon, results in a 1.5-2-fold increase of creatine synthesis from S-adenosylmethionine and guanidinacetic acid. An hour after glucagon administration to intact rats the GAMT activity of liver increases 9 times. The effect of glucagon is enhanced by insulin. Cycloheximide inhibits the increase of GAMT activity, induced by glucagon or a combination of glucagon and insulin. Experiments on tissue homogenates demonstrate that 3',5'-AMP in concentrations of 10(-8) --10(-2) M does not affect the GAMT activity or to some extent inhibits the enzyme. The homogenate incubation in a medium containing 10(-5) M epinephrine or 10(-7) M caffeine and 5 mM Mg2+ leads to an increase in the GAMT activity. Oligomycin removes the stimulating effects of caffeine and Mg2+ on the enzyme activation. This is probably due to the presence of 3',5'-AMP-dependent protein kinase in the mechanism of GAMT activation by cyclic AMP.


[The possible role of high molecular weight polyphosphates in chlortetracycline biosynthesis by Streptomyces aureofaciens].

The content of condensed inorganic polyphosphates in high productive and low productive Streptomyces aureofaciens strains has been determined. At all the stages of growing the quantity of these compounds in low productive strain is 8-10-fold higher than in high productive strains. Maximum accumulation of condensed inorganic polyphosphates in both the strains Str. aureofaciens corresponds to the end of logarithmic phase of growth. In both strains the presence of two enzymes of polyphosphate biosynthesis -- ATP:polyphosphate-phosphotransferase and 1.3-diphosphoglycerate:polyphosphate-phosphotransferase has been revealed as well as the presence of the enzymes of their utilisation -- polyphosphate:D-glucose-6-phosphotransferase, polyphosphate-phosphohydrolase, tripolyphosphosphate-phosphohydrolase and pyrophosphate-phosphohydrolase. The difference in the activity of 1.3 diphosphoglycerate: polyphosphate-phosphotransferase and polyphosphate-phosphohydrolase has been revealed in both strains studied. On the basis of the data obtained it is supposed that there is a possibility of using phosphorus and energy of high molecular inorganic polyphosphate in the biosynthesis of so called products of "secondary metabolism".


[Escherichia coli membrane-bound polyphosphatase].

A complex of polyphosphatase with E. coli membranes has been isolated and studied. It is shown by gel-filtration through G-200 Sephadex and centrifugation in sucrose concentration gradient that about 5% of polyphosphatase total content in cells is bound with the heterogenous fraction containing smooth membranes and the ribosome-membrane complex. On the basis of the data obtained it is suggested that the formation of a complex of polyphosphatase with membranes is a stage of synthesis and secretion of this enzyme to protoplasm.


[Changes in mitochondrial heterogenicity during aerobic growth of Saccharomyces cerevisiae yeasts].

Distribution of the activities of some mitochondrial enzymes after sucrose density gradient ultracentrifugation of cell homogenates of S. cerevisiae in the early and late exponential growth phases is studied. It is demonstrated that young yeast cells have a characteristic complex distribution of NADH oxidase (cyanide-sensitive), succinate:ferricyanide-oxidoreductase (or succinate:2,6-dichlorophenol indophenol-oxidoreductase), NADH:2,6-dichlorophenol indophenol-oxidoreductase and cytochrome oxidase activities in sucrose density gradient; the distribution patterns of these activities are different. All the above activities are detected in a single relatively narrow band in mature yeast cells. Similar results are obtained in the experiments with glucose or galactose as a carbon source in the yeast growth media. The Arrhenius plots for NADH oxidase (as well as for succinate:2,6-dichlorophenol indophenol-oxidoreductase) activity do not differ in the case of "light" and "heavy" mitochondrial structures characteristic of yeast cells in the early exponential growth phase. Nevertheless, "light" and "heavy" mitochondrial structures differ with respect of the arrangement of certain respiratory chain components in their membranes NADH-dehydrogenase and cytochrome oxidase). This conclusion is drawn from the results obtained in the study of the interaction of the two types of structures with Fe(CN)6(3-), a non-penetrating ion and the antiserum to yeast mitochondria.


[Interaction of adenosin-3',5'-cyclosulfate with adenosine-3'5'-cyclophosphate dependent protein kinase and phosphodiesterase].

Interaction of adenosine-3',5'-cyclosulphate (cAMS) cAMP analogue, having sulphur atom instead of phosphorus in a six-term cyclic system with pig brain proteinkinase and rabbit skeletal muscle phosphodiesterase is studied. The affinity of proteinkinase to cAMS was found to be in 25000 times lower than the affinity of cAMP, the affinity of cAMS to the active site of phosphodiesterase being high enough. It is suggested that in the regulatory subunit of proteinkinase positive kationic group participates in nucleotide binding by interacting with negative oxygen atom of six-term cyclophosphate system. There is no such a group in the active site of phospodiesterase, because the absence of negative charge in case of cAMS only slightly affects the constant of cAMS binding by phosphodiesterase.


[Potentiation of ACTH action by a fragment of it: a decrease in the rat adrenal ascorbic acid concentration in vivo].

The effect of tripeptide NH2CO-Arg-Pro-Val-NH2, the analogue of common fragments of some peptide hormones and kinins, on the content of ascorbic acid in rat adrenal glands in vivo is studied. It is shown that the peptide (5 divided by 3000 mkg/100 g body weight) produces no effect on the content of ascorbic acid in adrenal glands, but its injection half an hour before the administration of native ACTH canses a significant increase in the ACTH activity. The peptide possesses the myotropic activity; it exerts sinergism and potentiation of the ACTH action on rat ascending colon.


[Use of snake venom immobilized phosphodiesterase for analysis of the nucleotide composition of oligodesoxyribonucleotides].

A simple one-step technique for the analysis of nucleotide composition of oligodeoxyribonucleotides is described. The method is based on enzymatic hydrolysis and subsequent separation of hydrolysate on a "combined" column with Aminex A 28 anionite, on which top immobilyzed on DEAE cellulose snake venom phosphodiesterase is layed. The method is tested using a number of synthetic di- and tri-deoxyribonucleotides.


[Free radicals in the flavin reduction reaction. Interaction of reduced nicotinamide adenine dinucleotide with flavin in solution].

The study of the flavin mononucleotide (FMN)-reduced nicotinamide adeninedinucleotide (NADH) reaction was carried out both under aerobic and anaerobic conditions, using spectrophotometric and titrimetric methods. The consumption of NADH was shown to exceed two times the consumption of FMN in the anaerobic reaction and the rate constant in the aerobic reaction was found to be about 4 times of that of the anaerobic reaction. Moreover, the replacement of anaerobic conditions by aerobic ones at pH 5.0 resulted in a considerable increase of proton consumption rate in the reaction course. The data obtained are contradictory to the generally accepted hypothesis of hydrid-ion transfer in the reaction of NADH oxidation. It was assumed that this reaction proceeded through a homolytic pathway.


[Myeloperoxidase synthesis in white mouse leukocytes].

H3-leucine incorporation in myeloperoxidase (MPO) and into total soluble protein (TSP) from leucocytes of peritoneal exudate of mice was determined by immunological procedure. Maximal H3-leucine incorporation was observed within 48 hours and 16 hours of exposition for MPO and TSP respectively. Half-life period of MPO was 2,6 days which is comparable with the duration of neutrophils maturation up to the stage of metamyelocyte. Apparently, no essential formation of MPO takes place at the level of band and segmented neutrophils.


[Isolation and properties of carboxycathepsin (peptidyl-dipeptidase) from bovine lung tissue].

2200-fold purified homogenous preparation of carboxycathepsin (peptidyl-dipeptidase) is isolated from bovine lung. The enzyme isolated converts angiotensine I into angiotensine II and distroys bradikinin. It is active in neutral medium, is activated by chloride ion and is inhibited by EDTA and Middle Asian snakes venom. The molecular weight of the enzyme is 180 000-190 000 as estimated by means of polyacrylamide gel electrophoresis, its isoelectric point is 4.48-4.53. The comparison of properties and specificity of carboxycathepsin from bovine lung and kidney draws to the conclusion that both enzymes are identical.


Factors influencing the lipolytic response of swine (Sus domesticus) adipose tissue: ontogeny.

Swine adipose tissue was not responsive to glucagon at any age and only marginally to adrenocorticotropin (ACTH). The latter response could be greatly magnified and the sensitivity increased by addition of theophylline (Theo). The maximal lipolytic activity (MAX) obtained with ACTH plus Theo was minimal in the newborn, greatly increased in young animals, and reduced during the growing period; however, the sensitivity did not change. The MAX for Theo (plus low epinephrine) yielded a pattern similar to ACTH, but the sensitivity changed (most sensitive in younger animals). The MAX and sensitivity obtained with dibutyryl cyclic adenosine monophosphate were not appreciably changed during development.


Development of gluconeogenic enzymes in the newborn guinea pig.

The activities of key gluconeogenic enzymes in the liver of newborn guinea pigs delivered vaginally at term were monitored as a function of time following birth. The activities of glucose-6-phosphatase and fructose-1,6-diphosphatase did not show a significant increase over the first 72 h of life, neither did the activity of mitochondrial phosphoenolpyruvate carboxykinase. The mitochondrial enzyme pyruvate carboxylase and the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) both increased significantly in the first 24 h postpartum. Mitochondrial protein and succinate dehydrogenase activities showed only slight increases in the 72-hour period. Rapid depletion of liver glycogen was evident in these animals following birth, but severe hypoglycaemia was not evident. Mitochondrial and cytosolic PEPCK showed similar kinetic behaviour with respect to their affinities for oxalacetate and divalent metal cation Mn++, though the mitochondrial enzyme would accept Mg++ as the divalent metal in place of Mn++. The role of the compartmented PEPCK activities is discussed.


Ontogenic development of the responsiveness in cAMP synthesis to LH and PGE1 and gonadotropin receptors in the rat ovary.

Incubation of ovaries derived from rats aged 9, 13, 16, and 20 days with LH resulted in a significant rise in cAMP synthesis. The ovaries from 2- and 5-day-old rats were, however, unresponsive to stimulation by LH. By contrast, prostaglandin E1 (PGE1) evoked a sharp response in ovarian synthesis of cAMP even in 2-day-old rats. In order to determine why neonatal ovaries do not respond to LH, gonadotropic receptors have been studied. HCG binding sites were present in homogenates of untreated and gonadotropins primed rat ovaries. The specificity of this 125I-HCG binding was demonstrated by the competition of unlabeled HCG and LH for binding sites and failure of such binding to occur in specimens obtained from control tissues. FSH, ACTH and growth hormone were not able to compete significantly for 125I-HCG binding. The HCG receptor is present in ovaries during the whole postnatal period. The results suggest that the unresponsiveness of neonatal rat ovaries to LH is not caused by later maturation of gonadotropic receptors.


Recovery sleep following different visual conditions during total sleep deprivation in man.

The findings of visual impairment during total sleep deprivation were used as a basis for a possible link between vision and sleep. It was proposed that the level of visual load imposed during sleep deprivation was an important variable, and would have a substantial effect upon recovery sleep. Six young male subjects underwent two conditions of 64 h of sleep deprivation on separate occasions. One condition incorporated a high visual load, and the other a low load. Exercise and sound were balanced. All night sleep EEGs were taken for two baseline nights, and also for two recovery nights following each condition. There was a significant increase of stage 4 on all recovery nights and a REM rebound on the second recovery night. SWS, particularly stage 4, TST and REM density, were significantly greater following the high load. Implications of these findings for sleep theories and for sleep deprivation research are discussed.


Estimation of parameters for a model of transmitter release at synapses.

We consider models for the release of transmitter in response to nerve impulses, where it is assumed that quanta of transmitter are released from some of n sites, the probability of release from any site being p. It is assumed that the quantal size is either a constant or is distributed as a normal or a gamma variate. Observations on both spontaneous potentials and evoked potentials are used to obtain moment estimated of n and p. Large sample estimates of the standard errors of these estimates are given.


Establishment and characterization of an Epstein-Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt's lymphoma.

An Epstein-Barr virus (EBV)-negative lymphoblastoid cell line (LCL), BJA-B, was established from an African Burkitt's lymphoma (BL) which contained no detectable EBV DNA and did not express the EBV specified antigen EBNA, BJA-B cells grow in typically large, flat clumps. All carry surface-bound immunoglobulins, a B lymphocyte marker, and do not form rosettes with sheep erythrocytes. After infection of BJA-B cells by EBV the infected cells may produce either EBV-determined nuclear antigen (EBNA) or both EBNA and early antigen (EA), depending on the strain of EBV. The homogeneity of the BJA-B cell population with respect to immunological and isoenzyme markers and size suggests a clonal origin of the line. BJA-B is the first EBV-negative LCL established from an African Burkitt's lymphoma and demonstrates that an EBV-independent continuous B cell line can be established "in vitro" from other than leukemia or myeloma cells.


Renal transplantation and viral infections. II. Results of follow-up studies.

13 renal transplant recipeints were submitted to virological survey over a period of about 10 months following transplantation. They were compared to a group of individuals who underwent no transplantation : 7 patients on chronic hemodialysis and 10 healthy blood donors. The renal transplant recipients, showed rises in titer of antibodies against various viral antigens. Nevertheless, no other viral elimination but that of cytomegalovirus and herpes simplex virus was detected in them. Renal trnasplant recipients show significantly more often rises in virus antibody titers than the other individuals, when time of survey is taken into account. Some of these rises appeared to be simultaneous. Their mechanism is under debate : analysis of special cases and observation of associated immunohematological abnormalities are strongly suggesting that in some situations, rises in titer of viral antibodies may occur without infection.


Alterations in membrane-associated particle distribution during antidiuretic challenge in frog urinary bladder epithelium.

Frog urinary bladder epithelium has been examined by freeze-fracture electron microscopy of preparations previously fixed by glutaraldehyde either at rest or during antidiuretic challenge. All the agonists tested were observed to induce membrane particle clustering in the A face of the apical plasma membrane of granular cells. This was the case for the natural hormone (hypophysical extracts) and its presumed cellular mediator, adenosine 3',5'-monophosphate. Particle clustering was observed both in the presence and in the absence of water net flow and is thus independent of these movements. Clusters were also observed during hydrosmotic challenge by hypertonic serosal media, a condition which depresses transepithelial sodium transport. No complementary patterns of these A face clusters could be found on the B face. The significance of these membrane-associated particle clusters is discussed in terms of membrane structure and function.


Changes in the restriction of molecular rotational diffusion of water-soluble spin labels during fatty acid starvation of yeast.

Yeast mutants lacking fatty acid synthetase activity (fas-) die when deprived of saturated fatty acid under conditions which are otherwise growth-supporting. The spin label technique is used to show that restriction of molecular rotational diffusion of spin label molecules dissolved in aqueous zones increases several fold under conditions of fatty acid starvation while the apparent physical state of cellular hydrocarbon zones remains essentially unchanged. We focus attention on the cellular aqueous interior as the potential site of alteration under selective starvation conditions. Correspondences exist between restriction of molecular motion of water soluble spin labels dissolved in the cell and loss of cell viability. The correspondences to changes in the molecular motion of hydrocarbon soluble spin labels are much less or are not detectable.


Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time  taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.


The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of glass spheres.

In view of the advantages which are associated with the use of the BHK monolayer cell for the production of foot-and-mouth disease (FMD) virus, a unit system using glass spheres was developed to grow BHK monolayer cells and to test the susceptibility of such cells to FMD virus. The yield of cells and their susceptibility compares favorably with GREEN BHK monolayer cells which have been grown in Roux bottles.


The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of DEAE sephadex A50 beads.

Methods are described which make possible the production of foot-and-mouth disease (FMD) virus from BHK 21 C13 monolayer cells which have been grown on the surface of serum coated DEAE Sephadex A50 beads. The yield of cells and their susceptibility to infection by FMD virus are equivalent to conventional Roux monolayer systems. The potential for the commercial application of the DEAE Sephadex A50 system is discussed in relation to other unit process monolayer systems and in particular to the system in which cells are cultured in a deep bed of small glass spheres.


An etiologic and nosologic overview of craniosynostosis syndromes.

In the past, a great deal of confusion in the nosology of craniosynostosis syndromes has been apparent. Such syndromes should never be classified on the basis of which sutures are synostosed nor on the presence or absence of mental retardation. In this paper, they are classified on the basis of overall clinical similarity and genetic considerations. The findings in each syndrome, the genetic aspects and the problems in differential diagnosis are discussed. Disorders presented include the Kleeblattschädel anomaly, Crouzon syndrome, Apert syndrome, Pfeiffer syndrome, Saethre-Chotzen syndrome, Carpenter syndrome, Christian syndrome, Summitt syndrome, Baller-Gerold syndrome, Lowry syndrome, Gorlin-Chaudhry-Moss syndrome and three sporadic craniosynostosis syndromes. A discussion of spurious craniosynostosis syndrome entities is also presented.


[Half-life of rabbit heart mitochondria under normal conditions and in experimental allergic lesions of the heart].

Half-life of mitochondrial proteins and cytochrome C of rabbit myocardium was determined with glycine-I-C14 and H3-5-aminolevulinic acid, respectively, as precursors. Half-life of mitochondrial proteins calculated from the curve of the gradual radioactivity decline was 14.4 days, and that of cytochrome C--11.8 days; reutilization of glycine-C14 in plastic metabolism did not exceed 20%. No difference from control was noticed in rabbits with an experimental allergic heart damage.


[The biological role of estriol].

It was demonstrated on the uteri of women and guinea pigs that estriol (in vitro) possessed a marked affinity to the estradiol-binding system of human and guinea pig uteri; the activity of steroid-receptor interaction of estriol in vitro constituted 9.4% for guinea pigs and 17% for man in relation to the estradiol activity. Administration of estriol to guinea pigs in vivo in a dose of 0.25-0.5 mg led to a sharp reduction of the estradiol-binding capacity of the receptor system of the uterus. It is supposed that there existed a competitive relationship between estradiol and estriol for binding with the active centres of the receptor proteins of the uterus.


[Participation of the fibers of the lingual nerve in the transmission of mechanosensitive information from the tongue and oral cavity of the cat].

The role of the lingual nerve fibers in the transmission of mechanosensory information from the receptors of the tongue and oral cavity of a cat was studied. A chart of the afferent innervation of both mechanosensory surfaces of the tongue (dorsal and ventral surfaces) with fibers of the anterior medial and posterior branches of the lingual nerve was drawn up on the basis of the experimental material. Different ratios of the mechanoreceptor reactions were described.


[Plasma kinin system components of rabbits in acute inflammation].

During the peak of the acute aseptic inflammation of the subcutaneous connective tissue (on the 3rd-5th day) there occurred an activation of the plasma kinin system in the general circulation which expressed itself in an 1.5-2-fold increase in the activity of kallikreine, a 1.5-3-fold fall in the activity of kininase and in reduction by 25-26% of kininogen content. Thus, conditions were provided for the intesified formation and accumulation of free kinins participating in the second phase of permeability disorders of the vessels during the inflammation.


[Enzymatic activity in the osteoblasts of rabbits following combined trauma].

Experiments were conducted on young rabbits by the histochemical methods; the activity of NAD-, NADP-diaphorases, cytochromoxidase and alkaline phosphatase was studied in the osteoblasts 3, 6, 12, 24, 48 and 72 hours after combined radiation trauma. It appeared that a bone fracture led to an increase in the level of metabolic and energic processes in the osteoblasts as soon as in 12 hours. Irradiation of the animals depressed the activation (caused by the fracture) of all the enzymes investigated.


[Cyclic 3',5'-adenosine monophosphate stimulation of chloramphenicol-acetyltransferase synthesis in bacterial cellular systems].

A study was made of the effect of cyclic cyclic 3',5'-adenosinmonophosphate (c-AMP) on the production of chloramphenicol-acetyltranspherase (CAT) enzyme by whole bacterial cells in the strains of E. coli CSH-2/R222 and WZ-78/R222 (cya855). It was shown that CAT synthesis in the E. coli WZ-78/R222 strain was two times less in comparison with the E. coli CSH-2/R222 strain. C-AMP only insignificantly increased the production of CAT by the E. coli CSH-2/R222 strain, but considerably influenced the production of this enzyme by the WZ-78/R222 strain.


[Effect of morphine in vitro on the oxidative phosphorylation in rat liver mitochondria].

The rates of respiration in the presence of ADP and of phosphorylation as an ATP-ase activity of rat liver mitochondria was inhibited was in vitro by morphine with Ki=6.5 mM. The uncoupler-stimulated respiration of the mitochondria and the activity of ATP-ase and synthesis of ATP in the submitochondrial particles were not altered in the presence of morphine. It is suggested that morphine inhibited the adenine nucleotide transport through the mitochondrial membrane


[Ubiquinone content in albino rat kidney mitochondria following unilateral nephrectomy].

Following unilateral nephrectomy (in 72 hours) the weight of the remaining kidney increased by 23%, and the activity of succinic dehydrogenase--by 43.2%. The concentration of oubichinon in the hypertrophied kidney and its mitochondria showed no significant change for 3 days after the nephrectomy.


[Morphological and biochemical study of the organs of immunogenesis during the growth of established glioblastoma multiforme in albino rat brain].

Morphological changes in the lympho-reticular organs (the thymus, lymph nodes, spleen) were studied and the content of the RNA was determined by the biochemical method in 92 rats during the growth of the glioblastoma multiforme (strain 101/12) in the brain. The reaction of the organs of immunogenesis depended on the stage of the disease. On the 6th day after the transplantation of the tumour the increased activity of the epithelial-reticular portion of the thymus, hyperplasia of the follicles in the lymph nodes, and increased produciton of the RNA was observed. As to the tumour-bearing animals the destruction of the lymphoid elements and decreased synthesis of the RNA was noted. A giradual cell depletion of the spleen was revealed morphologically.


[Effect of qualitatively different carbohydrates on the lipoprotein spectrum and lipid composition of the blood in experimental animals].

Experiments on rats demonstrated that a high-carbohydrate diet with starch produced no changes in the lipoprotein composition and lipid concentration in the blood. Diets containing sucrose promoted an increase in triglycerides and of the pre-beta-lipoproteins. However, the effect of sucrose depended on the qualitative correlation of the carbohydrate and lipid components of the diet. A reduction of lipoproteins of low density and of the total cholesterol/phospholipid ratio on account of an increase of the latter in the blood was seen in rats given sucrose in the composition of the high-carbohydrate diet with a decreased amount of the saturated fat. Inclusion of sucrose into the diet with a physiological carbohydrate and lipid level (56 and 26% of total amount of calories, respectively) was accompanied by elevation of beta-lipoprotein level and an increase in the total cholesterol/phospholipid ratio.


[Role of bioenergetic changes in the mechanism of action of nonsteroid anti-inflammatory agents].

In experiments on rats the antiphlogistic action of sodium mephenaminate and salicylate was found to be more pronounced under conditions of ovalbumin than that of dextran inflammation. The antiphlogistic effect of sodium salicylate was greater than that of mephenaminate in ovalbumin inflammation; this correlated with a more marked drop in the content of lactic acid in the blood and with a more complete elimination of uncoupling of oxidative phosphorylation in the hepatic mitochondria than in case of mephenaminate injection. It is supposed that lowering the macroerg level by the principle of feed back action leads to intensification of phosphorylation; this provides elimination of inflammatory disturbances and can explain the antiphlogistic effect of the drugs.


A cholinoceptor antiserum: its pharmacological properties.

1 Sera of rabbits immunized against a nocotinic receptor-rich fraction, obtained from the electric organ of Torpedo marmorata, were tested for their pharmacological activity on different in vitro preparations. 2 Sera containing antibodies against the nicotinic receptor blocked neuromuscular transmission in the phrenic-nerve hemidiaphragm preparation without affecting the muscle responses evoked by direct electrical stimulation. Complement inactivated sera were still active. Immune sera, incubated for 15 min with a receptor-rich fraction, lost their activity. 3 The immune sera antagonized the responses elicited by acetylcholine on the frog rectus abdominis. 4 The immune sera tested in vitro decreased the compound action potential evoked in the superior cervical ganglion of the rat by electrical stimulation of the preganglionic nerve. 5 The sera did not show any activity on muscarinic receptors of the guinea-pig ileum preparation. 6 It is concluded that in sera obtained from immunized rabbits a substance is present with curare-like action, and that this activity is probably due to the presence of antibodies against the nicotinic receptor.


The reasons people give for taking overdoses.

This study investigates the reasons people give for taking overdoses of drugs. A representative sample of 128 subjects were interviewed immediately after their recovery from an overdose. During the interview they were given alternative reasons for taking overdoses and asked to choose any that applied to them. Spontaneous comments about suicidal intent were also recorded. Of the subjects, 44% indicated that they had wanted to die. On the basis of their choices 33% were 'seeking help', 42% 'escaping from the situation', 52% 'obtaining relief from a terrible state of mind' and 19% 'trying to influence someone'. The association between these various reasons and other expected effects or feelings associated with the act were examined by means of multidimensional scaling analysis. In addition, the characteristics of those expressing suicidal intent and other reasons were studied, together with such consequences as toxic effects and psychiatric after-care. The possibility is discussed that a large proportion of those indicating suicidal intent do so either to gain social acceptability for their act or to influence helping agencies.


Rectilinear scanning in the detection of acute myocardial infarction.

Myocardial scintigraphy with phosphate bone scanning agents is a new and very useful development in the detection of acute myocardial infarction. Initial experience using a rectilinear scanner is described in 50 consecutive patients admitted to hospital because of suspected myocardial infarction. The routine dose was 15 mCi/5 mg, 99Tcm stannous pyrophosphate intravenously with anterior, left anterior oblique, and lateral scans obtained 45-90 minutes after injection. There was only one false negative scan in 17 patients with proven acute myocardial infarction and that was 16 days after onset. There was no proven false positive investigation in seven patients in which fresh myocardial infarction could be definitely excluded, or in a further 11 cases in which it could be excluded with reasonable confidence. Myocardial scintigraphy was considered helpful in resolving the diagnostic problem in 27 out of 29 equivocal cases. It is concluded that myocardial scintigraphy with a rectilinear scanner is a highly accurate and safe procedure in the detection of acute myocardial infarction. The optimum scanning time is two to six days after onset of suspected infarction, when a diagnostic accuracy approaching 100 per cent can be expected.


Technetium-99m labelled imidodiphosphate: an improved bone-scanning radiopharmaceutical.

Technetium-99m labelled imidodiphosphate was prepared in the presence of stannous ions. It was evaluated as a bone-scanning agent in animals and patients. Comparative tissue distribution studies in mice showed a relatively higher uptake of radioactivity in bone when 99Tcm -labelled pyrophosphate and diphosphonate. Accumulation of radioactivity in soft tissues, especially kidneys, was less with this radiopharmaceutical. Results in patients were most satisfactory in delineating skeletal bone and identifying bone lesions with relatively small tracer doses of the radiopharmaceutical.


Nitrazepam: lastingly effective but trouble on withdrawal.

The sleep of 10 volunteers with an average age of 57 years was recorded electrophysiologically before, during, and after nitrazepam 5 mg nightly for 10 weeks. Sleep was longer and less broken on the drug and no tolerance was obvious after two months' use. Withdrawal of the drug, however, caused sleep to be temporarily worse than before the drug had been taken. Slow-wave sleep was reduced by nitrazepam, but the accompanying secretion of growth hormone was not impaired.


Attitudes and advice after myocardial infarction.

While medical opinion increasingly favours an active approach to rehabilitation after myocardial infarction, interviews with 40 patients and their relatives showed a low degree of understanding of medical advice and information. There was general satisfaction with treatment, but communication was seen as often being inadequate, vague, and conflicting.


Metabolic regulations of the rhythmic activity in pacemaker neurons. II. Metabolically induced conversions of beating to bursting pacemaker activity in isolated Aplysia neurons.

In pacemaker neurons of the sea hare Aplysia californica, isolated from their synaptic, ephaptic and humoral inputs, conversion of the regular beating to a bursting discharge pattern can be induced by certain cell metabolites. Administration of the phosphofructokinase (PFK) activator fructose-6-phosphate (F-6-P), or its nonmetabolizable analogue 1-deoxy-F-6-P, induced bursting discharges in R3, R5, R6 and R11 neurons, with spike doublets and triplets appearing transiently in the time pattern. With another PFK activitor, adenosine-5-monophosphate, only double spikes have been noted in R7, R8 and R14 neurons. Burst activity was induced also in the presence of the fructose-1,6-diphosphatase activators, citrate and 3-phosphoglycerate, in R9, R10 and R12 neurons. Cyclic 3',5'-AMP, which also activates the PFK (beside other effects on cellular metabolism), induced bursting discharges in all R3-R14 neurons. In contrast, the inhibitors of the PFK, citrate and ATP, decreased the spike activity of the bursting L3 and L6 neurons, even changing L3 neurons to the regular beating type. Among a variety of cell metabolites tested only pyruvate was able to induce a burst-like tendency in R9 neurons. The characteristic bursting patterns which appeared in the presence of the described metabolic effectors could not be duplicated by low Ca2+ and/or high K+ media nor by artificial shifts in membrane potential triggered by depolarizing and hyperpolarizing currents.


Effects of respiratory gases on cytochrome A in intact cerebral cortex: is there a critical Po2?

Changes in the redox level of cytochrome a and in the amount of oxygenated hemoglobin were measured by dual wavelength reflectance spectrophotometry in the intact cerebral cortex of cats (cerveau isolé preparation) and in unanesthetized rabbits with chronically implanted cranial windows. Increases in inspired oxygen were accompanied by an increase in the oxidation level of cytochrome a and an increase in the amount of oxygenated hemoglobin in the optical field. These changes were larger in the presence of 5% CO2. Reduction of the inspired oxygen concentration produced a decrease in the oxidation/reduction ratio of cytochrome a and a disoxygenation of hemoglobin. The presence of CO2 at these lower oxygen levels diminished the reduction of cytochrome a and the disoxygenation of hemoglobin. These data indicate that, in the resting subject, the reduction levels of cytochrome a are well above the low values seen in isolated mitochondria. They also indicate that the blood supply to the cerebral cortex is regulated at a level of slight hypoxia.


[Y-maze learning in two strains of mice. Effects of instrumental and pharmacologic sleep deprivation].

Effects of instrumental and pharmacological deprivation of sleep on Y-maze learning have been studied in two inbred strains of mice (C57BR/cd/Orl and C57BL/6/Orl), having identical sleep rhythms, but mainly differing in their ability to learn. Administration of alpha-methyl-DOPA (100 mg/kg) provokes complete suppression of paradoxical sleep (PS) for 9-11 h. Injection immediately after each training session over the first 5 days caused a delay in acquisition of an active avoidance task in C57BR mice. Treated C57BL/6 mice exhibited a significant facilitation of acquisition. Similar results were obtained by instrumental deprivation of sleep for 10 h.