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Biomedical subjects

M Esumi

Publications and source records attributed to M Esumi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Activation but not inactivation of the MTS1 gene is associated with primary colorectal carcinomas.

Several investigators have reported that alterations of the multiple tumor suppressor 1 (MTS1) gene frequently occurred in certain types of tumors. We examined the expression levels and somatic mutations of the MTS1 gene in primary colorectal carcinomas. The MTS1 transcript was quantitatively detected in 14 of 17 tumors, but in only one case of adjacent normal mucosa. Direct sequencing of the amplified MTS1 gene showed no somatic mutation in 17 primary colorectal carcinomas. These results indicate that inactivation of the MTS1 gene is not involved in pathogenesis of primary colorectal carcinomas, and that its enhanced expression is, instead, associated with primary colorectal carcinomas, especially markedly in the early stage of tumor progression.

Base Sequence↗
PubMed1996

Inactivation of hepatitis C virus cDNA transgene by hypermethylation in transgenic mice.

Transgenic mice were produced by microinjection of a partial hepatitis C virus (HCV) genome sequence including the structural protein region, under the control of the albumin promoter and enhancer into fertilized eggs of C57BL/6 and BDF1 mice. Three founders carrying at least five copies of the transgene but not expressing HCV-specific RNA were generated. Methylation analysis indicated that the transgene was extensively methylated. Mapping of methylated cytosine residues of the transgenic mouse DNA showed that all C residues of a particular part of the HCV genome but not all the CpG island like sequences were methylated. Transiently expressed HCV cDNA in COS7 cells and the active endogenous albumin gene were not methylated. Furthermore, 5-azacytidine, a potent demethylating agent, induced HCV gene expression in a line of these transgenic mice. These results suggest that methylation of HCV cDNA is a cause of its inactive expression in transgenic mice, and that this phenomenon may occur in other stable systems for expression of the HCV genome.

Animals↗
PubMed1996

Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system.

Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.

Amino Acid Sequence↗
PubMed1996

Murine humoral immune response against recombinant structural proteins of hepatitis C virus distinct from those of patients.

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti-E1 and anti-E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.

Animals↗
PubMed1995

Isolation and sequence polymorphism of a rat retinoblastoma (RB) cDNA.

A cDNA of the rat retinoblastoma gene (RB) was prepared from total RNA of rat liver using reverse transcription-polymerase chain reaction (RT-PCR). The 4432-nt sequence isolated contained 2700-nt translated and 1732-nt 3'-untranslated regions (UTR). The isolated cDNA detected poly(A)+RNAs of 5.4 and 3.4 kb in rat liver and kidney by Northern blot hybridization. The nt sequence of the isolated cDNA had 85% homology with that of mouse and 73% with human. The 899-amino-acid (aa) sequence was 95% homologous to that of mouse and 90% to human. The aa sequences of two functional domains of oncoprotein-binding and ten putative phosphorylation sites regulating RB function were conserved in the three species. However, the 3'-UTR were less homologous among the three, and had polymorphism in three portions, even in rats. These polymorphisms were strain-specific and genetically segregated. Thus, the rat RB cDNA and its sequence information may be useful for clarifying the role of the RB protein and genetic linkage analysis in basic biomedical research using rats, especially in experimental carcinogenesis.

Amino Acid Sequence↗
PubMed1995

[Microsatellite polymorphism of muscle glycogen synthase gene and non-insulin dependent diabetes mellitus].

It has been reported recently that A2 allele of muscle glycogen synthase gene determined by Xba I RFLP (restriction fragment length polymorphism) is related to the frequency of NIDDM in Finland. Using PCR-RFLP analysis, we detected no A2 allele in Japanese patients with NIDDM and control. We found a new microsatellite of CA repeat in the human muscle glycogen synthase gene, and the polymorphism of the repeat number was detected by the polymerase chain reaction. Six different alleles were observed, indicating the highly polymorphic nature of this marker. We analyzed the repeat numbers and the genotypes in diabetics and control. The allele frequencies were not significantly different between NIDDM and control. However, the allele frequencies in the patients without high total cholesterolemia was significantly different from those of control. These findings suggest that the muscle glycogen synthase gene or neighboring genes are related to one of the disease genes of diabetes mellitus without high total cholesterolemia.

Adolescent↗
PubMed1995

Biological characteristics of neuroblastoma with spontaneous tumor reduction: a case report.

The authors examined the biological characteristics of a neuroblastoma with spontaneous tumor reduction. A 6-month-old boy with a pelvic neuroblastoma underwent surgical extirpation of the tumor 1 month after diagnosis. The size of the tumor reduced spontaneously while he was awaiting operation. The low proliferative activity of the tumor cells and the presence of apoptosis in the tumor tissue were shown by an immunohistochemical method using anti-PCNA antibody and a DNA fragmentation analysis, respectively. These results suggest that the spontaneous tumor reduction seen in this patient may well be caused by the overwhelming apoptosis of tumor cells.

Apoptosis↗
PubMed1995

Defective copper binding to apo-ceruloplasmin in a rat model and patients with Wilson's disease.

To examine the mechanism of decrease in serum ceruloplasmin (Cp) in Long-Evans Cinnamon (LEC) rats, a proposed model of Wilson's disease, we analyzed Cp products at the stages of transcription and translation. Northern blot analysis and immunoblot analysis showed that the level and the molecular size of Cp mRNA and protein in LEC rats were similar to those in control Long-Evans-Agouti (LEA) rats. However, the ferroxidase activity of Cp was significantly decreased in LEC rats. We separated serum Cp into two forms by native polyacrylamide gel electrophoresis with pH modification: one was a holo-Cp with copper and ferroxidase activity, and the other was an inactive apo-Cp without copper. Holo-Cp was the predominant form in LEA rats and normal humans, whereas apo-Cp was the major form in LEC rats and patients with Wilson's disease. The cosegregation of apo-Cp predominance with the disease in LEC rats was analyzed using backcross rats. Apo-Cp was dominant in 8 of 11 offspring with disease but in none of 19 normal offspring. These results indicate that a genetic disturbance of copper binding to apo-Cp may be closely associated with the pathogenesis in LEC rats, and probably in Wilson's disease.

Animals↗
PubMed1995

A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins.

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.

Animals↗
PubMed1994

Sequence of the 5'-flanking region of the gene encoding human muscle glycogen synthase.

The 5'-flanking region of the gene encoding human muscle glycogen synthase was isolated from a human placental genomic library and sequenced. The sequence is TATA-less and G+C-rich, and putative transcription-controlling sequences were identified. Furthermore, a simple (dC-dA)n sequence repeat was identified about 4 kb upstream from the start codon. This sequence was highly polymorphic and five alleles were typed in the Japanese population using the polymerase chain reaction.

Animals↗
PubMed1994

Analysis of upstream region of hepatitis B virus core gene using in vitro transcription system.

Transcription of the core (C) gene of hepatitis B virus DNA (HBV-DNA) was studied by an in vitro transcription system using nuclear extracts of human liver cell (HepG2) and non-liver cell (HeLa) origins. RNA polymerase II-dependent run-off transcription of 3.5-kb (C) mRNA was observed in both nuclear extracts; but the efficiency was much higher in the HepG2 nuclear extract. Analysis of run-off transcripts using upstream deletion mutants of HBV-DNA demonstrated that there are two transcription start sites located at approximately nucleotide numbers (nt) 1,797 +/- 5 and 1,815 +/- 5. This analysis also suggested that the minimum core promoter sequence and a cis-acting and liver-specific element for C mRNA transcription are located in the downstream region from -80 and -110 (HincII site) of transcription start sites, respectively. DNA-binding protein assays using synthetic double-stranded oligonucleotide probes corresponding to three regions in the upstream region (nt from 1,401 to 1,760) of transcription start sites revealed that there are some liver cell-specific and non-specific DNA-binding proteins in both nuclear extracts. The amount of those proteins was generally higher in the HepG2 nuclear extract. However, no obvious correlation was observed in the present study between the presence of DNA-binding proteins and transcription activity of nuclear extracts in our system. The possible causes of this discrepancy are discussed.

Base Sequence↗
PubMed1994

Hepatitis C virus and liver diseases.

Hepatitis C virus (HCV) was identified molecularly and a procedure for its diagnosis was developed. In Japan, 70-80% of all cases of chronic liver disease, including hepatocellular carcinoma, are associated with HCV infection. Hepatitis C virus is a typical RNA virus with a high mutation rate. At least six variants of HCV have been identified by their nucleotide sequences. These variants are still classified into three types each containing at least two subtypes; that is, 1a (type I) and 1b (type II), 2a (type III) and 2b (type IV), and 3a (type V) and 3b (type VI). Type 1b (type II) is the predominant HCV in Japan. Even HCV cDNA clones isolated from a single patient showed mutations of HCV, especially in envelope-coding regions. Thus HCV may change during the course of chronic hepatitis due to the high mutation rate of HCV itself and elimination of some clones by immune reactions or interferon therapy. These findings explain the higher rate of chronic HCV infection and indicate that production of an effective vaccine is difficult.

Amino Acid Sequence↗
PubMed1994

Direct PCR of whole blood and hair shafts by microwave treatment.

We report a simple and rapid method for DNA preparation suitable for PCR by microwave irradiation. When mouse whole blood and hair shafts were directly irradiated and subjected to PCR, a unique gene such as the retinoblastoma susceptibility gene was reproducibly amplified. This method was useful for screening a transgene sequence in transgenic mice.

Animals↗
PubMed1993

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells.

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome.

The Japanese variant of the hepatitis C virus (HCV-N) genome, consisting of 9440 nucleotides in length, was cloned from a small amount (2 ml) of plasma from a single Japanese carrier by using RT-PCR and modified RT-PCR. The HCV-N genome has a long open reading frame that encodes a 3014 amino acid polyprotein with 340 and 57 bases of 5' and 3' non-coding sequences, respectively. HCV-N has a 4-amino-acid insertion in the NS5 region as compared to other HCV isolates, but this insertion is found to be very rare upon direct sequencing of that region. Comparative sequence analysis of all the complete and partial HCV sequences that were reported indicates that HCV can be subdivided into at least 4 groups. The HCV-N isolate has a high homology with HCV-J and HCV-BK (> 90%) and so belongs to group II, but shows less similarity to HCV-1 (> 78%, group I) and least to HC-J6 (> 67%, group III). Among these HCV isolates, the 5' non-coding region was the most conserved (> 93%) since it plays an important role in replication. The RT-PCR assay to detect HCV-RNA, using the primers deduced from this region, was very sensitive and specific. The putative core protein could become an important target for immunoassay because of a high degree of amino acid sequence similarity in that region. A high degree of diversity and a low similarity between each HCV isolate in the putative envelope protein play an important role in the chronicity of HCV infection and development of immunopreventive agents, such as immunoglobulin and vaccine for that infection.

Base Sequence↗
PubMed1993

A rapid molecular diagnosis of posttransfusion graft-versus-host disease by polymerase chain reaction.

A woman with recurrent Paget's disease of the vulva developed acute graft-versus-host disease (GVHD) 12 days after radical surgery and massive blood transfusion. Molecular diagnosis of lymphocyte chimerism in the peripheral blood was made by polymerase chain reaction (PCR) directed against a Y chromosome-specific sex-determining region Y (SRY) gene. PCR with skin biopsy after onset of GVHD also revealed infiltration of SRY-positive donor lymphocytes. The diagnosis was confirmed by HLA-DNA typing with PCR-sequence-specific oligonucleotide that revealed the presence of complex HLA-DR chimerism in the peripheral lymphocytes collected after onset of GVHD. The use of SRY-directed PCR is a rapid technique for the early diagnosis of acute posttransfusion GVHD in female patients.

Base Sequence↗
PubMed1992

Heterogeneity within the nonstructural protein 5-encoding region of hepatitis C viruses from a single patient.

Nucleotide (nt) sequence heterogeneity of the hepatitis C virus (HCV) genome derived from a single carrier was investigated. A polymerase chain reaction (PCR) product of 311 bp in the putative nonstructural protein 5-encoding region was directly sequenced, while part of a PCR product was cloned, and sequence analyses were carried out for 27 independent clones. Although 14 of the 27 clones were conserved, ten other types of nt sequences were found. The difference was at most 3 nt (1.1%). A directly determined sequence showed the major sequence of the cloned products. Since most of the nt changes occurred in the third letter of a codon, these nt changes might not have originated from random misincorporation during the PCR. These results of natural divergence of genome population in a single carrier suggest that HCV is a typical RNA virus with a quasi-species nature due to high mutation rates.

Base Sequence↗