Biomedical subjects

H Shirasawa

Publications and source records attributed to H Shirasawa.

At least 37 records · Page 2Linked to original sources

PubMed1997

Glutamate dehydrogenase mRNA is immediately induced after phencyclidine treatment in the rat brain.

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c-fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia.

Animals↗

PubMed1997

Transactivation of prothymosin alpha and c-myc promoters by human papillomavirus type 16 E6 protein.

The human papillomavirus type 16 E6 protein exerts a transforming activity through inactivation of tumor suppressor p53. Recently E6 has been shown to have additional transforming activities independent of p53. E6 is able to transactivate or repress several specific viral promoters. However, underlying molecular mechanisms and cellular target genes for the activity are not well understood. Using a differential hybridization technique, we identified the prothymosin alpha gene as a cellular target of E6 transactivation. E6 was able to transactivate the prothymosin alpha promoter in H358 cells lacking p53 and in C33A cells harboring a mutant p53 allele. Disruption of the E-box in intron 1 of the prothymosin alpha promoter abolished the responsiveness to E6. Then we determined if E6 up-regulates the expression of Myc, by which the prothymosin alpha promoter is transactivated through the E-box. We found that E6 is also able to transactivate the c-myc promoter in H358 cells and in C33A cells. These results suggest that E6 is able to transactivate the c-myc promoter independently of p53, and that the prothymosin alpha promoter is subsequently transactivated by Myc.

Animals↗

PubMed1997

Normal human fibroblasts immortalized by introduction of human papillomavirus type 16 (HPV-16) E6-E7 genes.

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only transfectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 microM ZnSO4, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.

Cell Culture Techniques↗

PubMed1997

Human papillomavirus type 16 E6 protein transcriptionally modulates fibronectin gene expression by induction of protein complexes binding to the cyclic AMP response element.

Although human papillomavirus type 16 (HPV16) E6 protein has a transcription-modulatory activity for a wide variety of viral promoters, a cellular target for this activity of E6 has not yet been identified. In this study, using differential hybridization, we identified a mouse fibronectin (FN) gene as a putative cellular target whose expression is up-regulated by E6. Chloramphenicol acetyltransferase (CAT) assays with mouse and rat FN promoter-CAT fusion constructs indicated that HPV16 E6 transactivates the FN promoters in a p53-independent manner. Deletion and site-specific mutation analyses revealed that transactivation by HPV16 E6 depends upon a cyclic AMP response element (CRE) located at -160 relative to the start site of transcription. Gel retardation assays demonstrated that nuclear extracts from the HPV16 E6-expressing cells, compared to those from parental 10T1/2 cells, have increased binding activity to the CRE. Antibodies against c-Jun and ATF-2 disrupted this binding activity. These data indicate that HPV16 E6 transcriptionally modulates FN gene expression via the CRE by inducing the binding of the protein complexes, probably including c-Jun and ATF-2, to the CRE.

Adenovirus E1A Proteins↗

PubMed1996

Expression, regulation and polymorphism of the mxi1 genes.

The myc proto-oncogene family plays an important role in the control of cellular growth and differentiation. Mxi1, one of the Max-associated proteins, has been known to have an antagonistic action on Myc activity. The mxi1 mRNA increased during growth inhibition and differentiation, and decreased with serum stimulation in mammal cell lines. We have also found an AAAAC polymorphic repeat in the 3' non-coding region of the human mxi1 cDNAs and a difference between the mxi1 mRNA half-lives in some different cell lines.

Base Sequence↗

PubMed1996

Isolation and serial propagation of human group C rotaviruses in a cell line (CaCo-2).

Rotaviruses were detected via electron microscopy in fecal specimens collected from school children during an outbreak of diarrhea and from a sporadic case in 1993 in Japan. All of the viruses were found to belong to human group C rotavirus by reverse passive hemagglutination assay (RPHA). These viruses replicated well in a human colon carcinoma (CaCo-2) cell line cultured in the presence of trypsin (4 micrograms/ml). This report demonstrates that human group C rotaviruses can be propagated efficiently in a cell line cultured in the presence of trypsin. The infected cells did not show any apparent cytopathic changes. However, virus was detected in the cell cytoplasm by immunofluorescence (IF) staining and in the culture supernatant by RPHA. On the basis of immune electron microscopy (IEM), virus particles collected from infected CaCo-2 cell cultures were confirmed to aggregate specifically with anti-human group C rotavirus antibody. The electrophoretic patterns of RNA segments extracted from viral particles found in the fecal specimens or infected cells were identical to those of human group C rotavirus. These results indicated that human group C rotaviruses were the causal agent of the diarrhea outbreak.

Caco-2 Cells↗

PubMed1996

Interleukin-6 (IL-6) production in carcinoma of the cervix.

Interleukin-6 (IL-6) is a pleiotropic cytokine that is not only a mediator in major immunologic reactions but also a growth factor of keratinocytes. We studied the IL-6 secretion in vitro of 15 human cell lines derived from both squamous cell carcinoma (SCC) and adenocarcinoma of the uterine cervix. Four of the eight well differentiated SCC secreted a large amount (> 1500 pg/48 h/10(6) cells) of IL-6 in nude mice. In contrast, poorly differentiated SCC cell lines and all of the 7 adenocarcinoma cell lines secreted a small amount (< 500 pg/48 h/10(6) cells of IL-6). The expression of IL-6 mRNA of the cell lines correlated well with their IL-6 secretion potential. However, the expression of IL-6 receptor did not correlate with the IL-6 secretory potential. We also studied the IL-6 secretion of freshly isolated normal squamous epithelium and of dysplastic epithelium. In culture, two normal squamous epithelia secreted a large amount (> 2000 pg/48 h/10(6) cells), whereas 8 dysplasia epithelia secreted an extremely small amount (< 10 pg/48 h/10(6) cells). About one-third of patients with SCC had a raised serum IL-6 value. IL-6 production may help to differentiate between SCC and adenocarcinoma of the uterine cervix. IL-6 regulation seems to change in the course of SCC carcinogenesis.

Adenocarcinoma↗

PubMed1996

Therapeutic effect of secretin in patients with jaundice; double-blind placebo-controlled multicentric trial.

Secretin, a gastrointestinal hormone, has been shown to have a potent choleretic effect. Having already obtained some beneficial effects with secretin in patients with intrahepatic cholestasis, we sought to confirm its effects in a double-blind placebo-controlled study in patients with mild jaundice after acute or during chronic hepatitis, where total bilirubin level was in excess of 4.0 mg/dl for 3 days or more. Patients with primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and familiar hyperbilirubinemia were excluded from the study. Ninety-three patients were included in this analysis, but the final evaluation covered 69 of them. No statistically significant differences were found in the reduction of serum bilirubin levels between secretin and placebo groups. As a number of patients with liver cirrhosis had been included, the subjects were subdivided into one group with cholestasis in hepatitis and one with liver cirrhosis. In the subgroup of cirrhotic patients who received secretin, serum levels of AST were significantly increased compared with the placebo group. However, since the choleretic effect of secretin is unique, further studies seem to be warranted.

Adolescent↗

PubMed1996

Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells.

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.

Animals↗

PubMed1996

p53-Dependent and -independent transactivation by the E6 protein of human papillomavirus type 16.

The mechanism by which the E6 protein of human papillomavirus type 16 (HPV-16) transactivates heterologous virus promoters has not been established. In this study, the involvement of p53-mediated transcriptional repression in transactivation by the HPV-16 E6 protein was examined using several virus promoters. HPV-16 E6 transactivated the TATA box-containing simian virus 40 early promoter and the Rous sarcoma virus long terminal repeat in p53-containing cells but not in p53-deficient cells. In contrast, the adenovirus E2 promoter was transactivated both in p53-containing and p53-deficient cells. These results indicate that the transactivation activity of the HPV-16 E6 protein is mediated by p53-dependent and promoter-specific p53-independent pathways.

Adenovirus E2 Proteins↗

PubMed1995

Cloning and sequencing of the murine farnesyltransferase alpha-encoding cDNA from a cell line which expresses the human papillomavirus type-16 E6 gene.

Using a differential hybridization technique, the murine farnesyltransferase alpha (FTA)-encoding cDNA was cloned from a mouse 10T1/2 cell line which expresses the human papillomavirus type 16 (HPV16) E6 gene. Sequence analysis revealed that the murine 1647-bp FTA cDNA encoded 377 amino acid (aa). The murine and human sequences showed 83.2% nucleotide and 92.6% aa sequence identity.

Amino Acid Sequence↗

PubMed1995

Cloning and sequencing of the murine Mxi1 cDNA.

The cloning and sequencing of the murine Mxi1 gene encoding Mxi1, one of Max-associated proteins, is described. Murine and human sequences showed 87.9% nucleotide (nt) and 90.3% amino acid (aa) sequence homology, whereas murine and zebra fish sequences showed 67.2% nt and 67.8% aa sequence homology.

Amino Acid Sequence↗

PubMed1995

Fc receptors in liver sinusoidal endothelial cells in NZB/W F1 lupus mice: a histological analysis using soluble immunoglobulin G-immune complexes and a monoclonal antibody (2.4G2).

In systemic lupus erythematosus accompanied by the abnormal appearance of circulating immune complexes (ICs), Fc gamma receptor (FcR)-mediated IC handling in macrophages including Kupffer cells has been shown previously. However, sinusoidal endothelial cells (SECs) largely ingest soluble immunoglobulin (Ig) G-ICs through FcRs. In this study, the character, antigenic expression, and activity (i.e., ligand-binding capacity of SEC FcRs in NZB/NZW F1 lupus and NZW nonautoimmune mice) were immunohistochemically analyzed using monoclonal antibody (MAb) 2.4G2 to FcRs and peroxidase-antiperoxidase IgG as a ligand on cryosections. MAb 2.4G2 stained SECs and blocked the ligand binding of SEC FcRs in both mice strains. The staining intensities with MAb 2.4G2 in SECs and the FcR activities in SECs alone and all sinusoidal cells in both mice strains reached their maximum values at the age of 5 months. Staining intensities in NZB/W F1 were significantly higher at 1 and 2 months and lower at 9 months than those in NZW. The number of Kupffer cells detected by MAb F4/80 to macrophages in both mice strains gradually increased until 5 months, but their number in NZB/W F1 at 9 months was twice as large as that in NZW. In conclusion, SEC FcRs in mice are low-affinity FcRs that react with MAb 2.4G2. The data of FcR activity suggest no impairment of the FcR-mediated IgG-IC binding on SECs in NZB/W F1 in early life.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Deletion in the L1 open reading frame of human papillomavirus type 6a genomes associated with recurrent laryngeal papilloma.

Human papillomavirus (HPV) type 6a genomes with a large deletion in their L1 open reading frames (ORF) were found in two of five recurrent cases of laryngeal papilloma. One of these mutant HPVs had a 186 base pair (bp) deletion near the N-terminus end of the L1 ORF, which encodes a major capsid protein. The other had a 454 bp deletion at the C-terminus end of L1 at which is located a nuclear localising signal (NLS). No other large deletion or insertion was found in the remaining regions of all five HPV6a genomes. The laryngeal papillomas which harboured the mutant viruses showed typical hyperplasia and pathological changes as observed in tumours induced by the wild-type virus. The biological significance of the two large deletions in the late region of HPV6a associated with laryngeal papilloma is discussed.

Amino Acid Sequence↗

PubMed1995

Intrasellar neuronal choristoma associated with growth hormone-producing pituitary adenoma containing amyloid deposits.

The histological, immunocytochemical, and ultrastructural features of an intrasellar neuronal choristoma associated with pituitary growth hormone (GH)-producing adenoma are reported. Immunohistochemistry studies and electron microscopy examination showed the adenoma cells to be positive for GH but negative for prolactin, and the neurons of the choristoma to have GH-releasing factor (GRF) neurosecretory activity. The adenoma also had many amyloid deposits in its extracellular space immunoreactive to GRF. This is the first report of the tumor containing amyloid deposits.

Adenoma↗

PubMed1995

A novel allelic variant of serum amyloid A, SAA1 gamma: genomic evidence, evolution, frequency, and implication as a risk factor for reactive systemic AA-amyloidosis.

Reactive systemic amyloidosis, also called AA-amyloidosis is a rare fatal complication of common chronic inflammatory diseases such as rheumatoid arthritis. It has been proposed that as yet undefined factors other than persistent elevation of serum level of the precursor protein, serum amyloid A (SAA), are also important for the development of AA-amyloidosis. In this work we show genomic evidence for a novel allelic variant of human SAA, SAA1 gamma, which we have recently identified at the protein level. The SAA1 gamma [Ala52(GCC), Ala57(GCG)] differed from SAA1 alpha [Val52(GTC), Ala57(GCG)] only at one base, indicating a single point mutation. On the other hand, SAA1 beta [Ala52(GCC), Val57(GTG)] had not only one, but additional differences in a nearby intron and this portion was identical to the SAA2 gene, suggesting a crossing-over between the SAA1 and SAA2 genes. Furthermore, we report that there was a significant difference in the observed numbers of SAA1 alleles between rheumatoid arthritis patients with AA-amyloidosis and the control population (chi 2(2) = 11.59, p = 0.003) with a higher frequency of gamma-allele in the AA-amyloid group (0.70 vs. 0.37). There was also a notable difference in the distribution of SAA1 genotypes (chi 5(2) = 14.63, p = 0.012) with an increased frequency of gamma/gamma-homozygotes in the AA-amyloid group (0.60 vs. 0.18). Thus our findings indicate that this novel allelic variant may be an important risk factor for the development of AA-amyloidosis.

Adult↗

PubMed1995

Rare association of human papillomavirus DNA with esophageal cancer in Japan.

To examine whether human papillomavirus (HPV) DNA is associated with esophageal cancer, frozen and paraffin-embedded neoplasms of the upper aerodigestive tract, including esophageal cancer, were investigated. DNA obtained from frozen specimens and cell lines were analyzed by both polymerase chain reaction (PCR) and Southern blot hybridization. DNA from paraffin-embedded samples were analyzed strictly by PCR. DNA of HPV types 6 and 11 was detected in papillomas of the upper respiratory tract at > 50%. However, HPV DNA was infrequently detected in specimens from the upper digestive tract (31 esophageal cancers and 2 esophageal carcinoma--derived cell lines), even by PCR at a sensitivity of 0.1 copy number per cell. These results suggest that the etiologic significance of HPV infection in esophageal cancer is negligible.

Base Sequence↗

PubMed1995

Isolation of influenza A and B viruses in HeLa cells.

The HeLa cell line which is one of the most popular cell lines was shown to be suitable for isolation of types A (H3N2) and B influenza viruses from throat washings of patients. Sixty-nine and 67 out of 147 throat washings taken from patients during the period from January to April 1994, were positive for influenza A virus in HeLa cells and MDCK cells respectively. Seven out of 10 throat washings taken between January and March, 1993, were positive for influenza B virus in MDCK. Of these 7, 4 were also positive for HeLa cells.

Animals↗