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Protection of Escherichia coli NIHJ and C57BL mice from the effects of 60Co gamma-rays provided by S-alk(en)yl-L-cysteines and their hydantoin derivatives was examined. E. coli (10(6) cells/ml) suspended in a 20 mM aqueous solution of one of the drugs was irradiated with 60 Gy of gamma-rays. Five week-old male mice were exposed to 5.0-9.5 Gy of gamma-rays after a single intraperitoneal injection of 0.75 mmol/kg body weight of each compound. In both E. coli and mice, S-allyl compounds afforded more effective radioprotection than S-propyl compounds. The replacement of the alpha-hydrogen of S-substituted cysteines by methyl groups decreased the radioprotective effect. Hydantoin derivatives were much more radioprotective than the original sulfur-containing amino acids. Especially, DL-5-allylthiomethyl-5-methylhydantoin had a remarkable radioprotective effect in mice. The gamma-radiolysis mechanism of thiomethylhydantoin derivatives was discussed in connection with the radioprotective effect of the drugs.

A new acidic ninhydrin method for determining free sialic acids is described. The method is based on the reaction of sialic acids with Gaitonde's acid ninhydrin reagent 2 which yields a stable color with an absorption maximum at 470 nm. The standard curve is linear in the range of 5 to 500 nmol of N-acetylneuraminic acid per 0.9 ml of reaction mixture. The reaction was specific only for sialic acids among the various sugars and sugar derivatives examined. Some interference of this method by cysteine, cystine and tryptophan was noted, although their absorption maxima differed from that of sialic acids. The interference by these amino acids was eliminated with the use of a small column of cation-exchange resin. The acidic ninhydrin method provides a simple and rapid method for the determination of free sialic acids in biological materials.

The enzyme activities involved in the transamination of L-cysteine sulfinate (L-alanine 3-sulfinic acid), L-aspartate and L-cysteine were examined in fetal, neonatal and maternal rat liver and placenta. In fetal and neonatal rat liver, aminotransferase activity was most active with L-cysteine sulfinate as a substrate and was also active with L-aspartate, while activity with L-cysteine was very low. The activity of transamination of L-cysteine sulfinate in rat liver developed in parallel with that of L-aspartate and L-cysteine. The aminotransferase activity markedly increased after the 19th day of gestation, reaching the same value as adult liver on the 3rd day after birth. The ratios of transamination of L-cysteine sulfinate to that of L-aspartate and to that of L-cysteine were constant during development. These observations suggest that L-cysteine sulfinate, L-aspartate and L-cysteine are transaminated by the same enzyme in the rat liver during development. Since placental aminotransferase activity was extremely low compared with that of the liver, it was suggested that the placenta did not play an important role in the transamination of these amino acids during pregnancy.

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.

Separation of the urinary ester-form bilirubin was attempted, and the results obtained may be summarized as follows: 1. A brown pigment was obtained from jaundiced urine by the following procedures; namely, salting out, methanol extraction, chloroform flocculation, and separation on cellulose column. The pigment has been found to be easily soluble in water, displaying the absorption maximum at 420 - 410 mμ at pH 7.0, and it also gave a positive reaction both to GMELIN's and EHRLICH's diazo reagents within a minute without the addition of alcohol. These characteristics agree well with those of the socalled ester-form bilirubin. 2. On the basis of the results of paper chromatography and paper electrophoresis, the pigment has been determined to contain no amino acid, steroid, nor reducing substance. Moreover, no glucuronic acid could be detected whether examined in vitro or by paper chromatography together with paper electrophoresis, either.

Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

Experimental beta-alaninuria was induced in rats by injection of (aminooxy)acetate (AOA), a potent inhibitor of aminotransferases, in order to elucidate the pathogenesis of hyper-beta-alaninemia. A 27-fold increase of beta-alanine (BALA) excretion was induced by subcutaneous injection of 1 5 mg of AOA per kg of body weight. A 13-fold and a 9-fold increase of beta-aminoisobutyric acid (BAIBA) and gamma-aminobutyric acid (GABA), respectively, were also induced simultaneously by the AOA injection. Identification of BALA and BAIBA isolated from the rat urine was performed by chromatographic and mass spectrometric analyses. The effects of AOA injection on the tissue levels of these amino acids were also studied. Contents of BALA in the liver and kidney and GABA in the brain increased significantly in response to AOA injection. The present study indicates that BALA transaminase is involved in hyper-beta-alaninemia.

Plasma amino acid abnormalities in rats treated with large doses of sake and whisky for 3 days were investigated under adequate nutritional conditions. A significant decrease in plasma branched-chain amino acid (BCAA) levels was observed in sake- but not whisky-treated rats. However, known factors affecting BCAA levels, such as serum insulin and plasma glucagon levels ahd BCAA-metabolizing enzyme (BCAA transaminase and branched chain alpha-ketoacid dehydrogenase) activities in the liver and skeletal muscle, were not significantly altered in the sake group. Furthermore, ethanol-metabolizing enzyme (alcohol and aldehyde dehydrogenases and the microsomal ethanol-oxidizing system) activities in the liver were not altered in the sake group. Other mechanisms need to be considered for explaining the diminished levels of plasma BCAA in sake-treated rats.

Branched chain amino acid (BCAA) transaminase activity increased in both the mitochondrial and supernatant fractions of brain from hepatic failure rats, in which a partial hepatectomy was performed 24h following carbon tetrachloride (CCl4) administration, although the activity of liver and skeletal muscle was the same as in control rats. The elevation of mitochondrial BCAA transaminase activity in liver-injured rats was partly due to increased activity of brain specific Type III isozyme. Branched chain alpha-ketoacid (BCKA) dehydrogenase in the brain homogenates was not significantly altered in acute hepatic failure rats, while the liver enzyme activity was markedly diminished. BCKA dehydrogenase activity in the brain homogenates was inhibited by adding ATP to the assay system, and was activated in vitro by preincubating the brain homogenate at 37 degrees C for 15 min. These findings suggest that brain BCAA catabolism is accelerated in acute hepatic failure rats.

Twelve patients were administered a vegetable protein-rich diet, which was low in methionine and high in the branched-chain amino acid (BCAA) to aromatic amino acid (AAA) molar ratio, and an animal protein-rich diet, high in methionine and low in the BCAA/AAA molar ratio. These diets were administered successively for one week each. Actually ingested amounts of tyrosine and methionine were significantly lower during the feeding of the vegetable protein-rich diet than the animal protein-rich diet. Serum methionine concentrations increased while on the animal protein-rich diet and decreased following the switch to the vegetable protein-rich diet. No other amino acid concentrations were affected. Significant differences were not observed in nitrogen balance or serum protein concentrations.

Fulminant hepatic failure (FHF) was produced in rats with intraperitoneal injection of D-galactosamine. Control rats received only physiological saline. 15N-leucine (200 mg/kg of body weight) was injected into the rats via the tail vein. Arterial blood was drawn before and 5, 15, 30 and 60 min after the injection of 15N-leucine. 15N-amino acids were determined quantitatively by gas chromatography and mass spectrometry. The plasma 15N-leucine level decreased logarithmically in the same manner in both groups. This result suggests that leucine is mainly metabolized in extrahepatic tissues. The incorporation of 15N into plasma isoleucine and valine was not significantly different between the groups. Plasma alanine and glutamine concentrations increased in controls and decreased in FHF rates after the injection. The incorporation of 15N into plasma alanine in rats with FHF was significantly later than in controls. This result may suggest that undergoing hyperammonemia causes to form more glutamine from glutamate in extrahepatic sites as the same manner as for chronic hepatic failure. Additionally, insulin levels increased temporarily after the injection of leucine in both groups. This increase may play a role in the decrease in plasma isoleucine and valine concentrations after injection of leucine.

Free amino acid contents in various guinea pig tissues were determined with an amino acid analyzer. The most abundant amino acids in these tissues were: Gly and Glu in the liver and kidney, Gln, Glu and Ala in the heart, Glu and Gln in the brain, Gly in the blood plasma and Lys in erythrocytes. Glutathione was present as the reduced form in these tissues. Cystine was not detected except in the blood plasma, but cysteine was present in these tissues. These results indicate that most thiols are present in the reduced form in these guinea pig tissues. Taurine contents were low compared with those in rat tissues. The results were discussed in relation to the metabolism of sulfur-containing amino acids, and it was suggested that the oxidative metabolism of L-cysteine was lower in guinea pig tissues than in rat tissues.

The author studied the distribution of polysaccharides and the amino-acid composition of cytoplasmic organellae, the problems that have come to call a great interest in the field of studies on cancer bearing animals. And also biochemical and electron microscopic observations were carried out to study the influences of cytoplasmic organellae in the cancer cells (AH 130), the livers of cancer bearing animals, and normal liver on the catalase activity of the liver. The results obtained are as follows : Cytoplasmic organellae of various cells do not affect so markedly the hexose metabolism of the liver. As for the amino-acid pattern of cytoplasmic organellae of various cells studied by paperchromatography, it is interesting to note that the pattern of the liver of cancer bearing animals, shows lack in histidine, while in can~er tissue and in the liver of cancer bearing animals an increase in phenylalanine can be observed. The decrease in the liver catalase activity is caused by the primary factor of cancer cells, especially their microsomes, and also by the secondary factor of the liver mitochondria in cancer bearing animals. On the other hand, the mitochondria of cancer cells, instead of reducing the catalase activity in the liver, markedly increases the catalase activity. By the morphological changes observed with light microscope and electron microscope, liver cells revealed marked morphological differences, proving that the microsomes of hepatoma cell induce considerably marked changes in the liver, while the mitochondria of hepatoma cell, on the contrary, induce the hypertrophy of liver cells. Sirriilarly in the electron microscopic observations the mitochondria of mouse liver injected with cancer mitochondria are enlarged, but no destruction of cellular structures such as cristae can be recognized. Also microbodies and the growing process of mitochondria can be observed, but no marked changes in endoplasmic reticulum.

With the purpose to see if GABOB is in any way concerned with the mechanism of the epileptic attack observations were carried on the oxygen consumption of the brain homogenates of rabbits, normal and CLA, and of human, epileptic and non-epileptic. The experiment proved that the oxygen consumption is increased in the epileptic brain and in the brain of CLA rabbit. It was raised by adding ATP-Na salt or DPN, but GABOB itself showed only a slight effect. The results suggested that the oxygen consumption of brain is not so closely correlated with GABOB, but there is a possibility that the decrease in GABOB contents in epileptic brain by the accelerated decomposition with its elevated oxygen consumption may be correlated to the epileptic attack, though the final conclusion requires further observations.

The biological specificity of GABOB on the blood pressure, respiration and body temperature was observed in dogs. The results show that GABOB has the similar action as GABA on the lowering of blood pressure as shown by subcutaneous, intravenous or intrathecal injection, but loses its action on the respiration. The specificity of GABOB action on the blood pressure is seen in the initiation of the effect at which the transient rise in pressure can be seen, the like of which can never be seen in the case of GABA injection. The lowering mechanism of GABOB on blood pressure should be the central one as the intrathecal injection is most effective comparing with those of intravenous and subcutaneous injection.

For the purpose to reveal the metabolic pathway of GABOB the analyses were performed with the GABOB containing fluid perfused through the liver and the brain of rabbits, and the following results were obtained. Qualitative observations by paperchromatography on the fluid containing GABOB after perfusing the organs proved the presence of some amino acids. These were identified as glycine, glutamic acid and glutamine. The observation on the GABOBcontaining fluid perfused the organs showed a decrease in GABOB and an increase in these amino acids. Quantitative observation proved a considerable increase in glycive and a moderate increase in glutamic acid and glutamine with a marked decrease in the amount of GABOB injected. From these results it is believed that GABOB is decomposed into glycine and acetic acid probably passing the stage of γ-aminoacetoacetic acid in one way and into glutamic acid by the transamination of GABOB with α-ketoglutaric acid in the other.

1. α-Aminoadipic acid and lysine are increased in the urine of thyroidectomized dog. 2. Pipecolic acid is increased in the urine of rat treated with thyroid extract. 3. Relation between thyroid function and lysine metabolism is discussed.

The effects of a low protein diet on the excretion of sulfate and taurine, major metabolites of L-cysteine in mammals, were studied in rats fed with synthetic 10% (group A) and 25% (group B) casein diets. The average excretions of total taurine (taurine plus hypotaurine) and total sulfate (free plus ester sulfate) (mumol/kg of body weight per day after the adaptation to the synthetic diet) in group A were 14.2 +/- 13.4 and 122.3 +/- 39.6, respectively, which were very low compared with 280.4 +/- 93.8 and 943.2 +/- 144.8, respectively, in group B. The taurine/sulfate ratio in group A was 0.12 +/- 0.11, which was significantly lower than that (0.30 +/- 0.08) in group B. A single intraperitoneal injection of 5 mmol of L-cysteine per kg of body weight in group A resulted in an increase in average taurine and sulfate excretion to 693.4 +/- 195.6 and 2440.6 +/- 270.0, respectively, and thus the average taurine/sulfate ratio increased to 0.29. These increases were transient and low taurine excretion resumed again 24 h after the L-cysteine administration. L-Cysteine injection in group B resulted in a similar increase in taurine and sulfate excretion, but the ratio changed only slightly (0.28). The present results suggest that in vivo production of taurine is reduced preferentially over sulfate production when sulfur amino acid supply is limited.

The present paper describes each pattern of the free amino acids in different parts of the dog brain determined by ion-exchange chromatography. The parts examined have been the cerebral cortex, cerebral white matter, cerebellar hemisphere, cerebellar vermis, caudate nucleus, thalamus, hypothalamus, and medulla oblongata. Gamma-aminobutyric acid concentration was the highest in the hypothalamus. Glutamic acid showed lower values in the white matter, hypothalamus, and medulla oblongata. Aspartic acid showed lower values in the white matter and caudate nucleus and higher values in the medulla oblongata. Glutathione and cystathionine showed higher values in the thalamus. N-Acetylaspartic acid showed lower values in the white matter and medulla oblongata. Glycine and alanine showed higher values in the medulla oblongata.

We administered a branched-chain amino acid (BCAA) infusion to 16 patients with hepatic failure and two healthy subjects, and then evaluated its effects on ammonia metabolism and amino acid metabolic pool. Immediately after the BCAA infusion, the venous blood ammonia concentration increased in 12 of 15 patients with hepatic failure and in both two healthy subjects. Glutamine (Gln) also rose in all cases following the BCAA infusion, and this rise was particularly marked in the hepatic failure group. The increase in Gln due to the BCAA infusion and the arteriovenous difference in the pre-administration ammonia concentration showed a good correlation. These results suggest an increase in glutamine cycle capacity in patients with hepatic failure.