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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 433 records · Page 24Linked to original sources
PubMed1987

Expression of human pancreatic secretory trypsin inhibitor in Saccharomyces cerevisiae.

Human pancreatic secretory trypsin inhibitor (PSTI) cDNA was expressed in Saccharomyces cerevisiae using the yeast acid phosphatase PHO5 promoter. The product encoded by the PSTI-coding cDNA was correctly processed in yeast cells, and the PSTI molecules were efficiently secreted into the medium. The amino acid composition and the N-terminal amino acid sequence of the secreted PSTI molecules were identical to those of the authentic PSTI polypeptides from human pancreas, and the product exhibited trypsin-inhibitory activity.

Acid Phosphatase↗
PubMed1987

Derivative spectrophotometric studies on cytotoxic effects of carbon monoxide.

Characteristics of cytochrome oxidase prepared from hearts of Sprague-Dawley male rats were studied with the use of the fourth derivative spectrophotometry in respect to the cytotoxic effects of carbon monoxide (CO). CO-exposed rats tended to show lower specific activities of cytochrome oxidase than control and recovered rats. Moreover, there was a significant difference in the fourth derivative spectral features of the enzyme: CO-exposed groups indicated peaks at 412 nm in the spectra while controls at 408 nm. This spectral difference seemed to reflect specific effect of CO on cytochrome oxidase, though such trace remained for not more than a day after death.

Animals↗
PubMed1987

Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

Carcinoma, Hepatocellular↗
PubMed1987

The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma.

Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. The distribution of DNA junctions along the virus genome suggests that there are recombination-proficient regions. Thus, about half of the integrants were the Coh type, viz., one of their virus-cell DNA junctions fell within the so-called cohesive end region that lies between two 11-bp direct repeats (DR1 and DR2) in the virus genome where transcription and replication of the genome are initiated. All the integrated virus genomes were defective at least in one site around the cohesive end region, particularly within the X gene. The recombination-proficient regions are used not only for formation of virus-cell but also of virus-virus junctions. Neither virus nor cell DNA show unique sequences at the junctions, and targets for integration lie on many different chromosomes.

Base Sequence↗
PubMed1987

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.

Cloning, Molecular↗
PubMed1987

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.

Animals↗
PubMed1987

Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.

Base Sequence↗
PubMed1987

The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck.

With v-yes DNA as the probe, a human cDNA library made from placental RNA was screened under relaxed conditions, and DNA clones derived from a novel genetic locus, termed lyn, were obtained. Nucleotide sequencing revealed that lyn could encode a novel tyrosine kinase that was very similar to mouse T-lymphocyte-specific tyrosine kinase p56lck and the v-yes protein as well as to the gene products of v-fgr and v-src. Northern hybridization analysis revealed that a 3.2-kilobase lyn mRNA was expressed in a variety of tissues of the human fetus. The pattern of lyn mRNA expression was different from those of related genes, such as yes and syn. Hybridization analysis of DNA from sorted chromosomes showed that the lyn gene is located on human chromosome 8 q13-qter.

Amino Acid Sequence↗
PubMed1987

Methylation of hepatitis B virus DNA and liver-specific suppression of RNA production in transgenic mouse.

Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB transgenic mouse carrying one copy of BamHI fragment of HBV with a deletion of Bg/II-Bg/II fragment, no RNA transcription was observed. Transgenic mice that had integrated either one or three tandem copies of HBV DNA produced RNA in various tissues except liver. However, neither initiation nor termination of transcription occurred at the correct place. Hepatitis B surface antigen and hepatitis B e antigen were not detectable in these transgenic mice. HBV DNA was methylated at all CCGG sequences, which might have resulted in aberrant RNA production. In two out of five transgenic mice HBV DNA was integrated into the Y chromosome. Although the frequency is unusually high, the mechanism is not known yet.

Animals↗
PubMed1987

[A novel oncogene from a human hepatocellular carcinoma].

Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assays using NIH3T3 cells and the calcium phosphate coprecipitation method. High-molecular-weight DNA obtained from the HCC tissues was employed for this purpose. One new transforming DNA, named lca (for liver cancer) was obtained, and its properties were studied in detail. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage. Restriction enzyme analyses showed that the minimum size of the lca DNA is about 10.5 kilobase pairs and that its nucleotide sequence is different from that of any known human gene or retroviral oncogene. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. It showed identical restriction enzyme cleavage profiles with its counterpart DNA obtained from normal tissue, indicating that there is no extensive DNA rearrangement associated with the activation process of the lca DNA. The normal counterpart DNA shows no transforming activity. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC.

Carcinoma, Hepatocellular↗
PubMed1987

Immunohistochemical studies of reflux nephropathy. The role of extracellular matrix, membrane attack complex, and immune cells in glomerular sclerosis.

Renal tissue obtained from 8 patients with reflux nephropathy was studied by immunofluorescence with well-defined mono- and polyclonal antibodies to extracellular matrix, intermediate filament proteins, membrane attack complex of complement (MAC), and immune cells. In both unscarred and scarred areas of the tissue, intrinsic components of the human glomeruli, such as Type IV collagen, laminin, and fibronectin, accumulated in the expanded mesangium of the glomeruli. Those components were diminished or absent in the hyalinized glomeruli. Type III collagen was occasionally localized to the mesangium and synechiae in the glomeruli and was frequently observed within the nearly hyalinized glomeruli. MAC was co-deposited with C3, C5, C9, and properdin in the sclerotic area of the glomeruli, along with a decrease in the reactivity of podocytes with anti-vimentin antibody. Numerous suppressor/cytotoxic T cells were identified in the glomeruli and widened interstitum. Monocytes/macrophages also infiltrated the glomeruli. The present study suggests that accumulation of extracellular matrix, activation of the complement system, and infiltration of suppressor/cytotoxic T cells and monocytes/macrophages are closely associated with the glomerular obsolescence and the progression of reflux nephropathy.

Adolescent↗
PubMed1986

Further evidence for beta-endorphin involvement in the long-lasting antagonistic effect of caerulein on amphetamine hyperactivity in rats.

The involvement of beta-endorphin (beta-END) in the long-lasting antagonistic effect of caerulein (CLN) on amphetamine (AMP) hyperactivity in rats was investigated. Injection of beta-END antiserum into the lateral ventricle or the nucleus accumbens abolished the CLN effect, whereas the injection of normal rabbit serum had no effect on the susceptibility to AMP for about 2 weeks, as observed in intact rats. Moreover, the CLN effect was blocked by long-term dexamethasone treatment, which inhibits beta-END synthesis. These findings indicate that beta-END in the nucleus accumbens plays an important role in producing the long-lasting effect on CLN, suggesting that dopamine release induced by AMP in the nucleus accumbens is presynaptically inhibited by opiate receptors activated by beta-END.

Amphetamine↗
PubMed1986

Chromosomal assignment of human genes for gastrin, thyrotropin (TSH)-beta subunit and C-erbB-2 by chromosome sorting combined with velocity sedimentation and Southern hybridization.

Human genes for gastrin, thyrotropin (THS)-beta subunit and c-erbB-2 were assigned to specific chromosomes using a single-laser cell sorter. For this purpose, condensed human chromosomes prepared from a karyotypically normal lymphoblastoid cell line were preliminarily fractionated by velocity sedimentation, and then sorted using a fluorescence-activated cell sorter. DNA was then extracted from the chromosomes, cleaved by restriction enzymes, and subjected to Southern hybridization using gene-specific radioactive probes. When the assignment of specific chromosomes was not possible due to chromosomal size overlapping, sorted chromosomes from cell lines carrying chromosomal translocation or from hybrid cells carrying known human chromosomes were used in addition. The results indicate that human genes for gastrin, TSH-beta, and c-erbB-2 are located on chromosomes 17, 1 and 17, respectively.

Cell Fractionation↗
PubMed1986

Study on the mechanism of the long-lasting antagonistic effect of caerulein on amphetamine-induced hyperactivity in rats.

The long-lasting antagonistic effect of caerulein (CLN) on amphetamine (AMP) hyperactivity was abolished in rats following partial 6-OHDA lesions of the nucleus accumbens (NA) or dopamine (DA) A10 area. CLN showed the long-term antagonistic effect on AMP hyperactivity in rats following complete 6-OHDA lesions of the DA A9 area. Neonatal monosodium L-glutamate-treated rats did not show the CLN effect. These results suggest that opiate receptors presynaptically located on DA neurons in the NA and some modulatory changes in the beta-endorphin system in the arcuate nucleus may play an important role in producing the CLN effect.

Amphetamine↗
PubMed1986

Primary structure of human salivary alpha-amylase gene.

A recombinant clone which covers the human salivary alpha-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary alpha-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene [Schibler et al., J. Mol. Biol. 155 (1982) 247-266].

Amino Acid Sequence↗
PubMed1986

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens.

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.

Amino Acid Sequence↗