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Biomedical subjects

A Ichihara

Publications and source records attributed to A Ichihara.

At least 127 records · Page 7Linked to original sources
PubMed1991

Hepatocyte growth factor in ascites from patients with cirrhosis.

Hepatocyte growth factor (HGF) stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in the ascites and plasma from patients with liver cirrhosis, but not in those from patients without cirrhosis. HGF was purified about 400-fold in 10% yield from cirrhotic ascites by ultrafiltration, cation-exchange chromatography on a S-Sepharose column, and affinity chromatography on a heparin-Sepharose CL-6B column. The partially purified factor was a heat- and acid-labile cationic protein with a molecular weight of 100,000-150,000. Its effect was half-maximal at 3.8 micrograms/ml, and was additive with those of insulin and epidermal growth factor. HGF in ascites from patients with cirrhosis had the same properties as HGF purified and characterized from rat platelets. These findings suggest that HGF is secreted into the ascites from the plasma or liver of patients with cirrhosis and may increase in the plasma with the development of hepatic impairment and act in repair of the damaged liver of patients with chronic liver disease.

Adult↗
PubMed1991

Induction of glutathione S-transferase P-form in primary cultured rat hepatocytes by epidermal growth factor and insulin.

The effects of epidermal growth factor (EGF) (10 ng/ml) and insulin (100 nM) on the expression of glutathione S-transferases (GSTs), especially the GST-P form (GST 7-7), were examined in primary cultured rat hepatocytes in serum-free medium. On culture with EGF and insulin, the GST activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were transiently decreased on day 2 to 10% of those of freshly isolated hepatocytes and then increased to 60 to 100% of those of freshly isolated cells on day 4. Western blot analysis of GSTs revealed that GST-P, which is not present in freshly isolated hepatocytes, was markedly induced and that GST subunits 3 and 4 of the Mu class also increased after addition of EGF and/or insulin, while the subunits 1 and 2 of the Alpha class disappeared. Northern blot analysis showed that on addition of EGF and insulin the level of GST-P mRNA was also elevated and expressions of the nuclear oncogenes c-jun and c-fos were enhanced. These results suggest that the enhanced expression of GST-P induced by EGF or insulin in primary cultured rat hepatocytes might be regulated by JUN and FOS proteins.

Animals↗
PubMed1991

Glucose regulation of growth hormone receptors in primary cultured rat hepatocytes.

The effects of nutritional factors on the regulation of GH receptors were studied by measuring the specific binding of 125I-human GH to rat hepatocytes cultured in various media. The binding of labeled GH to primary cultured hepatocytes decreased during culture, the extent of the decrease depending on the culture medium. The decreases of GH binding during culture in glucose-poor media (i.e. Williams' medium E, Dulbecco's modified minimum essential medium, and McCoy 5A medium) were significantly more than that in glucose-rich Waymouth's medium MB 752/1 (Waymouth's). In glucose-poor media supplemented with glucose, the extent of decrease of GH binding was comparable to that in Waymouth's. Other hexoses such as D-fructose and D-mannose also significantly reduced the decrease in GH binding. Scatchard plot analysis showed that the effect of glucose in preventing decrease of GH binding was due to change in the number of binding sites without significant change in their affinity. The effect of glucose was additive with that of dexamethasone, which induces GH receptors. Cycloheximide caused almost complete loss of GH binding by cells cultured in the presence of glucose. 2-Deoxyglucose reduced the effect of glucose, but did not affect that of dexamethasone. These results show that glucose counteracts decrease of GH binding sites during culture and suggest that it affects de novo synthesis of GH receptors, presumably through glycosylation of a certain protein that regulates GH receptor gene expression (new protein synthesis). The mechanism of the effect of glucose on induction of GH receptors seems to be different from that of dexamethasone.

Animals↗
PubMed1991

Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloride-treated rats.

A hepatocyte growth factor (HGF)-like substance that strongly stimulated DNA synthesis of adult rat hepatocytes in primary culture was found to increase markedly in liver of rats treated with carbon tetrachloride (CCl4). This increase of HGF-like activity was time- and dose-dependent, and 36 h after a dose of CCl4 of 0.2 ml per 100 g body weight the activity was about 20-times the normal level. The extent of induction of HGF-like factor correlated well with the extent of liver damage. The HGF-like factor was purified to homogeneity from the liver of CCl4-treated rats by a four-step procedure. The purified HGF-like factor had a molecular weight of 82-85 kDa, as estimated by SDS-PAGE, and was a heterodimer composed of a large subunit of about 69 kDa and a small subunit of 34 kDa linked by disulfide bridges. This factor had similar biological and chemical properties to HGF purified from rat platelets. Moreover, the N-terminal amino acid sequence of its 34-kDa subunit was identical to that of the small subunit of rat HGF. These findings indicate that the HGF-like factor in damaged liver of CCl4-treated rats is HGF and that liver itself can produce HGF when injured.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of the yeast proteasome PRS2 gene identical to the suppressor gene scl1+.

The YC7-alpha gene encoding a subunit of yeast proteasomes named PRS2 has been isolated and sequenced. Southern blot analysis after electrophoretic separation of yeast chromosomal DNAs showed that the PRS2 gene is located in chromosome VII, unlike the PRS1 gene for subunit YC1, is located on chromosome XV. Surprisingly, the overall structure of the PRS2 gene was found to be identical to that of the suppressor scl1+ gene restoring the SCL1-1 mutation that suppresses crl3 cycloheximide-resistant, temperature-sensitive lethality. The identity of these two genes indicates that the suppressive role of the scl1+ gene product on crl3 mutation could be attributed to proteasomal function.

Amino Acid Sequence↗
PubMed1990

Possible mechanism of nuclear translocation of proteasomes.

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co-localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X-X-K-K(R)-X-K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.

Amino Acid Sequence↗
PubMed1990

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells.

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.

Amino Acid Sequence↗
PubMed1990

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells.

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1990

cDNA cloning and sequencing of component C5 of proteasomes from rat hepatoma cells.

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide subunits. A cDNA for component C5 of rat proteasomes was isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The polypeptide deduced from the open reading frame consisted of 240 amino acid residues with a calculated molecular weight of 26,479. Computer analysis revealed little similarity of C5 to other proteins reported so far. The primary structure of C5 showed partial sequence homology to that of another component C3, but no regions of homology with the sequence of component C2. Thus C5 is concluded to be a new type of subunit of the proteasome complex.

Amino Acid Sequence↗
PubMed1990

The NH2-terminal residues of rat liver proteasome (multicatalytic proteinase complex) subunits, C2, C3 and C8, are N alpha-acetylated.

Rat liver proteasome (multicatalytic proteinase complex) is a 20S-ring shaped particle having a molecular mass of 750 kDa, and is composed of at least 13 non-identical components ranging from 21 to 31 kDa in size. We found here that the NH2-terminal residues of all the known 13 components, except for C5, are not reactive to phenylisothiocyanate. Among them, components C2, C3 and C8 are blocked in their NH2-termini with N alpha-acetyl-Met, N alpha-acetyl-Ala, and N alpha-acetyl-Ser, respectively. The NH2-terminal portions of C2, C3, and C8 exhibit sequence similarity to one another, but that of the non-blocked component C5 differs from those of C2, C3, and C8.

Acetylation↗
PubMed1990

Molecular cloning of cDNA for proteasomes from rat liver: primary structure of component C3 with a possible tyrosine phosphorylation site.

Proteasomes are multicatalytic proteinase complexes consisting of multiple components. Previously, we reported the cloning and sequencing of cDNA for the largest component, C2, of rat liver proteasomes [Fujiwara, T., Tanaka, K., Kumatori, A., Shin, S., Yoshimura, T., Ichihara, A., Tokunaga, F., Aruga, R., Iwanaga, S., Kakizuka, A., & Nakanishi, S. (1989) Biochemistry 28, 7332-7340]. In the present study, the nucleotide sequence of another component (C3) of proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The deduced sequence of component C3 consists of 234 amino acid residues with a calculated molecular weight of 25,925. These values are consistent with those obtained by protein chemical analyses. A single mRNA species hybridizing to the C3 cDNA of rat liver was expressed in all rat tissues examined and in a variety of other eukaryotic organisms, its distribution being similar to that of C2 mRNA. The wide distribution of the gene product, possibly C3, suggests that this protein functions similarly in most eukaryotes. C3 is an unmodified protein of a single gene and differs from any other known protein, but its overall amino acid sequence resembles those of other proteasomal components, including C2, suggesting that these components belong to a single family of proteins with the same evolutionary origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver.

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.

Age Factors↗
PubMed1990

Inhibition of protein phosphatases by microcystins and nodularin associated with hepatotoxicity.

Microcystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent "activation" of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds, alpha-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

Animals↗
PubMed1990

Abnormally high expression of proteasomes in human leukemic cells.

Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells.

Antibodies, Monoclonal↗
PubMed1990

Organization and structure of the 5' flanking region of the rat serine dehydratase gene.

For study of the mechanisms regulating the induction of serine dehydratase by various hormones in rat liver [Noda et al. (1988) J. Biol. Chem. 263, 14764-14768], we cloned the gene for this enzyme from a rat genomic library. The gene spans about 7.5 kilobases and consists of nine exons and eight introns. The exon-intron boundaries are consistent with the "GT-AG" rule. Southern blot analysis of rat genomic DNA suggested the presence of one copy of serine dehydratase gene per haploid genome. The 5' end of serine dehydratase mRNA is located 148 nucleotides upstream of the initiator methionine codon. ATG, determined by primer extension analysis and S1 nuclease mapping, although an alternative transcription initiation site(s) may be located a few bases downstream. The 5' flanking region of the gene lacks typical TATA and CCAAT sequences, but contains AATAAA and CATT sequences, at -25 to -20 and -54 to -51, respectively. Furthermore, there are five GC box-related sequences. There are three putative glucocorticoid-responsive elements and two copies of the CGTCA motif of the cAMP-responsive element upstream of the promoters. The 5' flanking sequence shows more than 98% homology with that reported by Ogawa et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5809-5813], but the first exons have different sequences. Another difference is that a segment of 108 nucleotides is located just upstream of exon 5 in the sequence reported here, but is included as an exon in the sequence of Ogawa et al. The possibility to producing two species of serine dehydratase mRNAs from a single gene by transcription from different sites and alternative splicing is discussed.

Animals↗
PubMed1989

Separation of yeast proteasome subunits. Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli.

On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5. The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution. Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli. These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti.

Adenosine Triphosphatases↗
PubMed1989

Molecular cloning of cDNA for proteasomes (multicatalytic proteinase complexes) from rat liver: primary structure of the largest component (C2).

Proteasomes (multicatalytic proteinase complexes) from rat liver are composed of at least 13 nonidentical components [Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, M., Sato, M., Tanaka, N., Katsube, Y., Kameyama, K., & Takagi, T. (1988) J. Mol. Biol. 203, 985-996]. The nucleotide sequence of one major component (C2) of the proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a mixture of synthetic deoxyribonucleotides as a probe. The sequence was composed of 1174 nucleotides including a coding region for the entire protein and noncoding regions of both the 5'- and 3'-sides. The polypeptide deduced from the open reading frame consisted of 263 amino acid residues, and its molecular weight was calculated to be 29,516. The partial amino acid sequences of several fragments (approximately 45% of the total residues), which were obtained by cleavage of C2 with lysyl endopeptidase and cyanogen bromide, were determined by automated Edman degradation and found to be in complete accordance with those deduced from the cDNA sequence. The amino acid composition of C2, determined by chemical analysis, was also consistent with that deduced from the cDNA sequence, indicating that the cloned cDNA actually encoded component C2. Computer analysis revealed little structural similarity of C2 to other proteins reported so far. Northern blot hybridization analyses showed that the mRNA encoding this novel protein C2 was expressed in all the rat tissues examined and in a variety of eukaryotic organisms such as amphibia, birds, and mammals with slight species-specific differences in size.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗