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Biomedical subjects

A Ichihara

Publications and source records attributed to A Ichihara.

At least 235 records · Page 13Linked to original sources
PubMed1979

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture.

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.

Alkaline Phosphatase↗
PubMed1978

Purification and properties of L-lysine-alpha-ketoglutarate reductase from rat liver mitochondria.

L-Lysine-alpha-ketoglutarate reductase (N5-(1,3-dicarboxypropyl)-L-lysine: NADP+ oxidoreductase (L-lysine-forming, EC 1.5.1.8) was purified from rat liver mitochondria to a homogeneous state judged by SDS polyacrylamide gel electrophoresis, and its molecular weight was estimated as 52000. On Sepharose 4B filtration it has a molecular weight of 230 000 and it is suggested that the active enzyme is a tetramer of subunits of similar size. The purified enzyme was clearly separated from saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine:NAD+ oxidoreductase (L-glutamate-forming, EC 1.5.1.9). The reactions of purified L-lysine-alpha-ketoglutarate reductase favored the forward reaction (saccharopine formation) and the rate of the reverse reaction (lysine formation) was only 3--5% that of the forward reaction. The forward reaction was specific for L-lysine, alpha-ketoglutarate and NADPH and followed Michaelis-Menten kinetics, whereas the dose vs. response curve of the reverse reaction was sigmoidal with saccharopine. Among the amino acids examined, ornithine, leucine and tryptophan inhibited the forward reaction competitively. These results are different from earlier reports on human and yeast enzymes. The fact that rats fed on lysine-deficient diet do not lose weight much is discussed in relation to the properties of this enzyme.

Animals↗
PubMed1978

Inhibition of protein synthesis in mouse L cells by poly-L-ornithine.

A snythetic polypeptide, poly-L-ornithine (pLo; average molecular weight, 13,000), inhibited protein synthesis in mouse L cells in suspension culture. The inhibition was dependent upon both the concentration of pLo and the cell density, and the dose for half-inhibition was correlated with the cell density. Cell viability remained high in the concentration range of PLo causing inhibition of protein synthesis. This inhibition was prevented by addiction of calf serum with pLo, but not by washing the treated cells with serum or a high concentration of salt. PLo had no effect on protein synthesis in a cell-free system prepared from L cells or rabbit reticulocytes. The polysome profiles of cells treated with pLo were similar to those of control cells. Experiments on the effect of interchange of the ribosomes and supernatants of control and pLo-treated cells showed that the ribosomes from pLo-treated cells were inactive. The inactivation of these ribosomes was partially prevented by the presence of 120 mM K+ in the medium during pLo treatment. The inhibition of protein synthesis by pLo, therefore, may result from the binding of pLo to the cell membranes, causing leakage of intracellular K+ and thus inactivating ribosomes.

Blood↗
PubMed1978

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis.

Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5 X 10(7) and 1.0 X 10(8) cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions or freshly isolated cells and cultured cells were compared in this medium. In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated. These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.

Animals↗
PubMed1977

Induction of ornithine decarboxylase in cultured mouse L cells. II. Effects of additions of amino acids and serum.

Induction of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells by components of the culture medium was investigated. It was found that further addition of amino acid mixture, but not of calf serum, to confluent cells of 5 day culture induced ODC and that this induction was accelerated by actinactinomycin D. In salt solution, addition of either amino acids or serum alone did not cause full induction, but addition of both together did. This induction, in contrast, was inhibited by actinomycin D. Induction by insulin, but not by cyclic AMP, was enhanced by a higher concentration of amino acids. These results can be explained by supposing that in non-growing cells there is stable RNA which is involved in ODC induction, possibly mRNA of ODC, and that the observed induction is caused by inhibition of enzyme degradation and accelerated translation, while in growing cells this RNA is unstable and ODC induction is controlled at the level of transcription.

Amino Acids↗
PubMed1977

Isozyme patterns of branched-chain amino acid transaminase in human tissues and tumors.

The isozymes (enzymes I and III) of branched-chain amino acid transaminase (EC 2.6.1.42) from various human tissues were separated by DEAE-cellulose column chromatography. Their distributions were found to be considerably different from those in rat tissues reported before. In rats, all tissues examined contained enzyme I and in addition enzyme II was found in the liver and enzyme III in the brain, ovary, and placenta. In humans, however, enzyme II was not found in any tissues examined including the liver, and enzyme III was found in many other tissues besides brain, ovary, and placenta. All normal human tissues except the lung, ovary, and brain contain less enzyme III than enzyme I. Various human cancers of the liver, kidney, stomach, pancreas, and uterus showed significantly higher ratios of enzyme III to enzyme I than those of the corresponding normal tissues. Fetal liver and kidney also contained much higher concentrations of enzyme III than adult liver and kidney. These findings suggest that change of the isozyme pattern of this enzyme in cancer is similar in humans and rats, and that cancer tissues tend to express more immature phenotypes than normal tissues. Enzymes I and III of human tissues showed the same substrate specificities for valine, leucine, and isoleucine, and these amino acids competed for the active site of the enzyme. Thus the hereditary diseases, hypervalinemia and hyperisoleucine-leucinemia, may be due to genetic alteration of the enzyme protein, resulting in change of its substrate specificity.

Animals↗
PubMed1976

Transaminase of branched chain amino acids. XI. Leucine (methionine) transaminase of rat liver mitochondria.

An aminotransferase (transaminase) which is active for leucine and methionine, but not for valine or isoleucine, was purified from rat liver mitochondria. The purified preparation appeared homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was shown to be 55 000 by gel filtration. It differed from enzyme II (leucine aminotransferase, EC 2.6.1.6) in the supernatant fraction, another transaminase which is also specific for leucine and methionine, in molecular weight, Km values for substrates, electrophoretic mobility, chromatographic behavior and heat stability. From comparison with related transaminases it was concluded to be a new enzyme and named mitochondrial leucine (methionine) transaminase.

Animals↗
PubMed1976

Induction of ornithine decarboxylase in cultured mouse L cells. I. Effects of cellular growth, hormones, and actinomycin D.

The activity of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells in the confluent state was induced within 4 hr by cyclic AMP (cAMP) or by insulin. During growth of L cells the concentration of cAMP increased first, then induction of ODC occurred and finally the cell number increased: the levels of cAMP and ODC increased only transitorily and returned to the basal levels when the cells become confluent. In growing cultures, however, the presence of cAMP reduced induction of ODC and cell growth. These results suggest that cAMP is involved in induction of ODC and that its concentration may be important for enzyme induction as well as for cell growth. Actinomycin D with or without these inducers stimulated induction of ODC in L cells, whereas cycloheximide inhibited it, suggesting that these hormones affect the translational level of ODC synthesis. The effect of actinomycin D on induction of ODC was much greater in non-growing cells than in growing cells. It was also found that the half life of ODC was 81 min in non-growing cells and 112 min in growing cells. This suggests that turnover of the enzyme is more rapid in the non-growing than in the growing state and that there may be an RNA fraction which controls its turnover and which also has a very short half life.

Animals↗
PubMed1976

Control of ketogenesis from amino acids. IV. Tissue specificity in oxidation of leucine, tyrosine, and lysine.

In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In in vitro studies the abilities of slices of various tissues of rats to form 14CO2 from 14C-amino acids were examined. With liver, but not kidney slices, addition of alpha-ketoglutarate was required for the maximum activities with these amino acids. Among the various tissues tested, kidney had the highest activity for lysine oxidation, followed by liver; other tissues showed very low activity. Kidney also had the highest activity for leucine oxidation, followed by diaphragm; liver and adipose tissue had lower activities. Liver had the highest activity for tyrosine oxidation, but kidney also showed considerable activity; other tissues had negligible activity. In in vivo studies the blood flow through the liver or kidney was stopped by ligation of the blood vessels. Then labeled amino acids were injected and recovery of radioactivity in respiratory 14CO2 was measured. In contrast to results with slices, no difference was found in the respiratory 14CO2 when the renal blood vessels were or were not ligated. On the contrary ligation of the hepatic vessels suppressed the oxidations of lysine and tyrosine completely and that of leucine partially. Thus in vivo, lysine and tyrosine seem to be metabolized mainly in the liver, whereas leucine is metabolized mostly in extrahepatic tissues and partly in liver. Use of tissue slices seems to be of only limited value in elucidating the metabolisms of these amino acids.

Adipose Tissue↗
PubMed1976

Further studies on the isozyme patterns of branched-chain amino acid transminase in cultured rat hepatocytes.

Among several lines of cultured rat hepatocytes examined, one line, IAR-22, contained enzyme III as well as enzyme I of the isozymes of branched-chain amino acid transaminase (EC 2.6.1.42) and this cell line was tumorigenic. None of the cell lines tested contained enzyme II (EC 2.6.1.6) or tyrosine transaminase (EC 2.6.1.5) and addition of dexamethasone did not induce these transaminases. The PC-1 line, containing only enzyme I and having a high percentage of diploid cells, showed deviation of its chromosomal number and tumorigenicity, but no change in its isozyme pattern when transformed with N-methyl-N'-nitro-N-nitrosoguanidine. Therefore, judging from expression of isozymes of the transaminase, this transformed cell line retained moderate differentiation.

Amino Acids↗
PubMed1976

Isozyme patterns of branched-chain amino acid transminase in cultured Morris hepatoma 7316A.

Cultured cells from Morris hepatoma 7316A contained isozymes I and II, but not isozyme III, of branched-chain amino acid transaminase. They also contained tyrosine transaminase. Isozyme II and tyrosine transaminase were induced by addition of cortisol. These findings agree well with in vivo findings. However, prolonged culutre of the cells for over 500 days caused deviation of the chromosomal bumber and activity of both enzymes, and they were no longer affected by cortisol. A tumor formed by back-transplantation of the cells showed the typical isozyme pattern of rapidly growing hepatomas, such as Yoshida ascites hepatomas, i.e., isozymes I and III. These results were discussed in relation to change of gene expression during culture.

Animals↗