Biomedical subjects

A Ichihara

Publications and source records attributed to A Ichihara.

At least 145 records · Page 8Linked to original sources

PubMed1989

Na+, K+-specific inhibition of protein and peptide hydrolyses by proteasomes from human hepatoma tissues.

Proteasomes were purified from human hepatoma tissues, and their sensitivities to Na+ and K+ were examined. At concentrations of 10 mM or more, these cations were found to inhibit completely polylysine-activated casein degradation by the purified proteasomes. They also strongly inhibited the hydrolyses of peptides, although to a lesser extent. On the other hand, they reversed the inhibitory and stimulatory effects of polylysine on the hydrolyses of Suc-Leu-Tyr-AMC and Cbz-Ala-Arg-Arg-MNA, respectively. These results suggest that Na+ and/or K+ may be involved in the regulation of intracellular protein breakdown by controlling the multicatalytic activity of proteasomes.

Calcium↗

PubMed1989

Half-life of proteasomes (multiprotease complexes) in rat liver.

Proteasomes (large multicatalytic proteinase complexes) are abundant in rat liver, constituting approximately 1.0% of the total soluble proteins. In the present study, the apparent half-life of the proteasomes was determined to be 12-15 days from the decay curve of isotopically labeled enzymes in vivo, suggesting their slow turnover. This finding together with the ubiquitous distribution of proteasomes in eukaryotic cells (Tanaka et al., 1988 J. Biol. Chem. 263, 16209-16217) indicates that proteasomes belong to a family of proteins with house-keeping function.

Animals↗

PubMed1989

Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific functions.

When hepatocytes isolated from adult rats were cultured in the presence of 10 mM nicotinamide, insulin- and epidermal growth factor-induced DNA synthesis and cell proliferation were found to be greatly stimulated, and the cells were able to be kept alive for more than one month. In the nicotinamide-treated hepatocytes, albumin and tryptophan 2,3-dioxygenase mRNAs were present at much higher levels than in the untreated control, and the inducibility of tryptophan oxygenase gene expression by dexamethasone and glucagon was also preserved. Without nicotinamide, primary cultured hepatocytes were viable for only 5-7 days and the hepatocyte-specific phenotypes were rapidly lost. The intracellular NAD level was maintained in the nicotinamide-treated hepatocytes at or above the level in intact liver but depleted in hepatocytes without nicotinamide. These results suggest that the maintenance of the intracellular NAD level is essential for the growth and functioning of hepatocytes and that nicotinamide can preserve the NAD level by blocking NAD degradation as well as by acting as a precursor for NAD synthesis.

Albumins↗

PubMed1989

Autodegradation of rat liver proteasomes (large multicatalytic proteinase complexes).

Purified proteasomes (large multicatalytic proteinase complexes) were found to be very stable, showing no change in activities or structures during prolonged incubation in medium of pH 7.5 at 37 degrees C. However, on addition of urea they were degraded autocatalytically in a time- and dose-dependent manner, suggesting that destruction of the proteasomal complexes acts as a signal for their autolysis. ATP at a physiological concentration greatly stimulated the urea-dependent breakdown of proteasomes. The autolysis induced by urea was almost completely inhibited by hemin, but not by other protease inhibitors tested, such as leupeptin, chymostation and Ep-475. Thus, autolytic degradation of proteasomes appears to be important for the regulation of enzyme levels in eukaryotic cells.

Adenosine Triphosphate↗

PubMed1989

One of two subunits of masking protein in latent TGF-beta is a part of pro-TGF-beta.

A high molecular mass latent form of transforming growth factor type-beta (TGF-beta) was purified to homogeneity from rat platelets by a seven-step procedure involving group-specific affinity chromatographies on Red-Toyopearl and zinc chelating-Sepharose. The purified latent TGF-beta was a complex of TGF-beta (25 kDa) and the binding protein previously named masking protein (approximately 400 kDa) [(1986) Biochem. Biophys. Res. Commun. 141, 176-184]. Analysis of the peptide structure by gel electrophoresis showed that the masking protein consisted of two subunits of 39 kDa and 105-120 kDa linked by disulfide bonds. N-terminal amino-acid sequencing of the 39 kDa subunit indicated that this subunit was identical to the N-terminal part of the TGF-beta precursor.

Amino Acid Sequence↗

PubMed1989

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (multiprotease complexes) in liver.

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.

Animals↗

PubMed1989

Involvement of the proteasome in various degradative processes in mammalian cells.

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.

Adenosine Triphosphate↗

PubMed1989

Purification and structural analysis of a latent form of transforming growth factor-beta from rat platelets.

A latent form of transforming growth factor type-beta (TGF-beta) with a high molecular weight was purified to homogeneity from rat platelets by a six-step procedure. The yield of the purified latent TGF-beta from platelets of 2,500 rats was 1.4 mg. The purified latent TGF-beta was activated by treatment with urea at concentrations of over 4M or acidic solutions of below pH 4. SDS-PAGE and gel filtration chromatography showed that the latent TGF-beta consisted of active TGF-beta and glycoproteins of about 200 kDa as masking components, and that under physiological conditions, these components formed a high molecular weight complex of about 400 kDa linked by non-covalent bonds. Here, we found that the masking protein was composed of one large subunit of about 110 kDa and two small subunits of 39 kDa linked by disulfide bridges. The N-terminal amino acid sequence of the small subunit was identical to the N-terminal region of the TGF-beta precursor lacking a signal peptide. From these findings, we proposed a structural model for the latent TGF-beta from rat platelets.

Amino Acid Sequence↗

PubMed1989

Role of substrate in reversible activation of proteasomes (multi-protease complexes) by sodium dodecyl sulfate.

Previously, we reported that proteasomes (large multi-protease complexes) are present in a latent state in a variety of eukaryotic cells, and can be activated by treatment with various compounds such as sodium dodecyl sulfate (SDS) or poly-lysine (Tanaka et al. (1988) J. Biol. Chem. 263, 16209-16217). In the present study, the mechanism of activation of latent proteasomes by SDS was examined. Latent proteasomes were greatly activated by addition of low concentrations of 0.04 to 0.08% SDS in the presence of substrate. This activation appeared to be reversible, because SDS-activated proteasomes returned to a latent state when the concentration of SDS was reduced by dilution. In contrast, in the absence of substrate, latent proteasomes lost their activity almost completely in an irreversible fashion within a few minutes during treatment with SDS at either 0 or 37 degrees C. Interestingly, SDS-treated proteasomes were markedly protected against this rapid inactivation by either a peptide or protein substrate. Moreover, removal of the substrate after activation of proteasomes caused their rapid irreversible inactivation. These results indicate that the substrate is necessary for reversible activation of latent proteasomes by SDS. This effect of substrate is presumably important in regulation of intracellular protein breakdown by activated proteasomes in eukaryotic cells.

Cysteine Endopeptidases↗

PubMed1989

[Growth and differentiation of neonatal rat hepatocytes in primary culture].

Liver plays a central role in metabolism of the body and it regenerates actively after injury. Primary cultured hepatocytes are suitable for studies on its growth and complex functions, because these cells mimic many liver functions and respond well to various hormones just like hepatocytes in vivo. Adult hepatocytes in culture are quiescent, but they proliferate in the presence of appropriate mitogens. Rat platelets contain a hepatocyte growth factor (HGF) and growth inhibitors. This HGF was found to be a labile protein of 100 kDa consisting of subunits of 34 and 69 kDa. One inhibitory factor, identified as transforming growth factor (TGF)-beta, shown to be released from platelets in a latent form and to be activated by its dissociation from bound masking proteins. Interleukin (IL)-1 and -6 also inhibit growth of hepatocytes. Besides these humoral factors, we found that the cell density in culture is an important regulatory factor for hepatocyte growth; a high cell density suppresses cell growth and promotes expression of differentiated liver functions. The roles of these humoral and membranous factors in the mechanism of liver regeneration are discussed. In contrast, neonatal hepatocytes in culture can grow autonomously without an added mitogen and they secrete several growth factors into the medium. This autocrine growth ceases rapidly after birth and differentiated characters appear in neonatal hepatocytes in culture. This change from growth to differentiation of immature hepatocytes can be stimulated by contact of the cells with adult hepatocytes. Therefore, micro-architecture of surroundings of hepatocytes is very important for regulation of liver functions and growth of not only adult hepatocytes, but also neonatal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells.

Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared. They showed latency in breakdown of [methyl-3H]casein, but were greatly activated in various ways, such as by addition of polylysine. They all degraded three types of fluorogenic oligopeptides at the carboxyl side of basic, neutral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, suggesting that they had three distinct active sites. The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region. Their physiochemical properties, such as their sedimentation coefficients (19 S to 22 S), diffusion coefficients (2.0-2.6 X 10(-7) cm2 s-1), molecular masses (700-900 kDa), and circular dichroic spectra, were similar. Their amino acid compositions were also very similar. Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center. All the proteasomes seemed to be multisubunit complexes consisting of 15-20 polypeptides with molecular masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity. Moreover, they differed immunologically, as shown by Ouchterlony tests and immunoblotting analyses, although cross-immunoreactivities of some subunits or domains were observed. These results indicate that the sizes and shapes of these proteasomes have been highly conserved during evolution, but that they show species-specific differences in immunoreactivities and subunit structures. Thus proteasomes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms ranging from man to yeast. This distribution implies the general importance of these proteasomes for proteolysis.

Amino Acids↗

PubMed1988

Molecular organization of a high molecular weight multi-protease complex from rat liver.

A latent multifunctional protease with a molecular weight of 722,000 to 760,000 purified from rat liver cytosol has been reported. This paper reports on the structure and subunit composition of the enzyme. Electron microscopy showed that the enzyme was a ring-shaped particle of 160(+/- 7) A diameter and 110(+/- 10) A height with a small hole of 10 to 30 A diameter (1 A = 0.1 nm). Small-angle X-ray scattering analysis indicated that the enzyme had a prolate ellipsoidal structure with an ellipsoid cavity in the center. The maximum dimension of the enzyme was estimated to be 210 A from a pair-distance distribution function. The radius of gyration obtained from a Guinier plot and the Stokes radius based on the ellipsoidal model were 66 A and 76 A, respectively. On two-dimensional gel electrophoresis, the purified enzyme separated into 13 to 15 characteristic components with molecular weights of 22,000 to 33,000 and isoelectric points of 4 to 9. These multiple components were not artifacts produced by limited proteolysis during purification of the enzyme, because the cell-free translation products in a reticulocyte lysate with poly(A)-mRNA of rat liver consisted of multiple components of similar sizes, and because peptide mapping analyses with lysylendopeptidase and V8 protease demonstrated clear differences in the primary structures of these components. The 13 main components were isolated from the purified enzyme by reverse-phase high performance liquid chromatography and shown to be non-identical. A model of the enzyme is proposed on the basis of these observations and previous physicochemical studies. Interestingly, the morphology of this protease is similar to that of the 16 to 22 S ring-shaped particles found in a variety of eukaryotic organisms. The structural similarity between this multi-protease complex and various reported subcellular particles is discussed.

Animals↗

PubMed1988

Requirements of both glucocorticoids and glucagon as co-inducers for activation of transcription of the serine dehydratase gene in cultured rat hepatocytes.

Induction of translatable mRNA for serine dehydratase (SDH) in primary cultured rat hepatocytes requires both dexamethasone and glucagon or cAMP (Noda, C., Tomomura, M., Nakamura, T., and Ichihara, A. (1986) J. Biochem. (Tokyo) 95, 37-45). This unique hormone requirement was studied further with a cDNA probe complementary to SDH mRNA in primary cultured hepatocytes of adult rats. Dot-blot hybridization analysis of RNA showed that SDH mRNA was induced by dexamethasone and glucagon together, but not by either alone. Insulin or epinephrine caused 40% inhibition of this induction of SDH mRNA. Cycloheximide prevented the induction of SDH mRNA by dexamethasone and glucagon, suggesting that ongoing protein synthesis is required for the induction by glucocorticoids and glucagon. In vitro transcription experiments using nuclei isolated from cultured hepatocytes showed that transcription of the SDH gene was not affected by either dexamethasone or glucagon alone, but markedly enhanced by both hormones together, and that this enhancement was inhibited by insulin or epinephrine. These results indicate that the inhibition of SDH induction by epinephrine or insulin was due to effects of these hormones on the transcriptional rate of the SDH gene.

Animals↗

PubMed1988

Involvement of proteasomes (multicatalytic proteinase) in ATP-dependent proteolysis in rat reticulocyte extracts.

The role of proteasomes, particles with latent multicatalytic proteinase, in ATP-dependent proteolysis in rat reticulocyte extracts was examined. Removal of proteasomes from the extracts by immunoprecipitation caused almost complete inhibition of ATP-dependent degradation of [3H]methylcasein, without affecting ATP-dependent proteolysis. Peptide fragments of [3H]casein, obtained by cyanogen bromide cleavage, were rapidly degraded in an ATP-independent fashion and this activity was not affected by removal of the proteasomes. These results suggest that proteasomes are involved in ATP-dependent proteolysis in the extracts and that they catalyze the initial cleavage of large proteins.

Adenosine Triphosphate↗

PubMed1988

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence.

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.

Amino Acid Sequence↗

PubMed1988

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture.

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.

Animals↗

PubMed1988

Effect of modifying agents on the phenotypic expression of cytochrome P-450, glutathione S-transferase molecular forms, microsomal epoxide hydrolase, glucose-6-phosphate dehydrogenase and gamma-glutamyltranspeptidase in rat liver preneoplastic lesions.

The expression of A and P forms of glutathione S-transferase (GST-A and P), two cytochrome P-450 isoenzymes (P-450 PB3a and P-450 MC2), microsomal epoxide hydrolase (mEHb), glucose-6-phosphate dehydrogenase (G6PD) and gamma-glutamyltranspeptidase (gamma-GT) was compared in preneoplastic liver lesions and background parenchyma of F344 rats post-treated with butylated hydroxyanisole (BHA), ethoxyquin (EQ) or acetaminophen (AAP). These latter three compounds have been shown to inhibit hepatocarcinogenesis after initial treatment with N-ethyl-N-hydroxyethylnitrosamine (EHEN) and a significant decrease in the number of enzyme-altered foci and nodules positive for GST-P, GST-A, G6PD and gamma-GT and negative for P-450 PB3a, P-450 MC2 was associated with their administration. Whereas in the foci case the decrease was most prominent for non-discrete (heterogeneous) type lesions, the results of quantitation of nodules revealed a most significant alteration in the discrete homogeneously staining population. This indicates that BHA, EQ and AAP have the potential to inhibit the growth of the phenotypically stable lesions thought most likely to be the immediate precursors of hepatocellular carcinomas. The two anti-oxidants were associated with periportal increase of all enzymes investigated, whereas AAP induced GST species and mEHb in the perivenular zone. Irrespective of slightly elevated enzyme levels in surrounding parenchyma, mEHb antibody binding levels within lesions showed a reciprocal shift from positive to negative in rats treated with BHA, EQ and AAP.

Animals↗

PubMed1988

Autocrine mechanism of growth of neonatal rat hepatocytes in primary culture.

Hepatocytes from neonatal rats of 0 to 3 days old grew actively in primary culture without added serum or growth factors. In these culture conditions, growth of hepatocytes decreased progressively with increase in age of the rats from which they were isolated, and hepatocytes from rats of 2 weeks old showed scarcely any growth. Actively growing hepatocytes were found to secrete a growth factor that promoted their growth and that of Swiss 3T3 cells, but not that of adult hepatocytes. This growth factor in conditioned medium of growing hepatocytes was heat- and acid-stable, but sensitive to trypsin, and had a molecular weight of over 10,000. It did not inhibit the binding of [125I]epidermal growth factor to its receptor, and its growth promoting activity was not inhibited by monoclonal antibody against insulin-like growth factor II. Therefore, it seems to be a new growth factor. These results, together with previous findings (Nakamura, T., Nagao, M., & Ichihara, A. (1987) Exp. Cell Res. 169, 1-14) demonstrated a reciprocal relation between growth and maturation of neonatal hepatocytes during development, like that of adult cells, but indicated that unlike growth of the latter, growth of neonatal cells is induced by an autocrine mechanism.

Aging↗