Biomedical subjects

M Yutsudo

Publications and source records attributed to M Yutsudo.

At least 37 records · Page 2Linked to original sources

PubMed1996

Isolation of a novel gene down-regulated by v-src.

We have isolated a novel gene which was expressed in normal rat cells, but completely suppressed in cells transformed by v-src. The molecularly cloned cDNA was about 1.8 kb in size, containing an open reading frame composed of 464 amino acid residues. DNA sequence analysis showed that there was no corresponding gene in the data bases. Besides the suppression of gene expression in the v-src transformed cells, its expression was also strongly suppressed in cells transformed by other oncogenes such as v-abl, v-fps, v-mos, v-sis, v-K-ras, and polyomavirus middle T, but not affected in cells transformed by human papillomavirus type 16 E6E7 and polyomavirus large T. We named the gene drs for a gene down-regulated by v-src.

Amino Acid Sequence↗

PubMed1996

Involvement of Epstein-Barr virus expression in testicular tumors.

PURPOSE: Because orchitis has been described as a symptom of infectious mononucleosis which is caused by Epstein-Barr virus (EBV), a human tumor virus, we tried to ascertain the relationship between EBV and testicular tumors. MATERIALS AND METHODS: Sixteen seminomas, 11 embryonal carcinomas and 25 nonmalignant control testes were examined for persistence and expression of the EBV genome. To detect expression of EBV, mRNA in situ hybridization and immunofluorescence staining by monoclonal antibodies were performed. To confirm the EBV genome in testes, we used nested polymerase chain reaction (PCR). RESULTS: Messenger RNA in situ hybridization showed that all 27 seminomas and embryonal carcinomas expressed EB viral RNA, but the 25 nonmalignant testes did not. Monoclonal antibody staining showed EBV-related nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1 expression in testicular tumors. Nested polymerase chain reaction detected the EBV genome in normal testes as well as in testicular tumors. CONCLUSIONS: These results suggest that EBV is related to testicular tumors.

Antigens, Viral↗

PubMed1996

Immunophenotypic features and configuration of immunoglobulin genes in hairy cell leukemia-Japanese variant.

Immunophenotypes and Ig gene rearrangements were investigated in 12 patients with a variant form of hairy cell leukemia (HCL) termed HCL-Japanese variant (HCL-J), and in an HCL-J-derived cell line. The leukemic cells of HCL-J characteristically showed the phenotype of CD20+, CD5-, CD10-, CD11c+, CD22+, CD24- and CD25-. Ig light (L) chain was undetected in nine cases, and the remaining four cases expressed kappa chain. Expression of Ig heavy (H) chain was studied in nine cases. In addition to Igkappa+ cases showing expression of predominantly gamma H chain isotype, alpha chain was detected in one case without expression of L chain. Rearranged bands in Ig heavy chain (JH) genes were recognized in all 12 cases tested. Rearranged bands in kappa chain genes and germline configuration in chi chain genes were seen in all three Igkappa+ cases tested. Four of nine cases without expression of L chain had a rearranged chi chain gene. The other three cases had chi chain genes in the germline configuration and rearranged and/or deleted kappa chain genes. In the remaining two cases, no rearrangement in either kappa or chi chain genes was detected. The Ig gene configuration and expression in HCL-J, partially overlapping with those described for immature B cell leukemia, were dissociated from the cytological features and CD20+, membrane CD22+ phenotype characteristic of mature B cells.

Antibodies, Monoclonal↗

PubMed1995

Cloning of a new cytokine that induces IFN-gamma production by T cells.

The mechanism underlying the differentiation of CD4+ T cells into functionally distinct subsets (Th1 and Th2) is incompletely understood, and hitherto unidentified cytokines may be required for the functional maturation of these cells. Here we report the cloning of a recently identified IFN-gamma-inducing factor (IGIF) that augments natural killer (NK) activity in spleen cells. The gene encodes a precursor protein of 192 amino acids and a mature protein of 157 amino acids, which have no obvious similarities to any peptide in the databases. Messenger RNAs for IGIF and interleukin-12 (IL-12) are readily detected in Kupffer cells and activated macrophages. Recombinant IGIF induces IFN-gamma more potently than does IL-12, apparently through a separate pathway. Administration of anti-IGIF antibodies prevents liver damage in mice inoculated with Propionibacterium acnes and challenged with lipopolysaccharide, which induces toxic shock. IGIF may be involved in the development of Th1 cells and also in mechanisms of tissue injury in inflammatory reactions.

Amino Acid Sequence↗

PubMed1995

Tumorigenicity of EBNA2-transfected cells.

The Epstein-Barr virus nuclear antigen 2 (EBNA2) gene is thought to be important for transformation by Epstein-Barr virus (EBV), but the mechanism of this transformation is little understood. Here, to examine the transforming ability of EBNA2, we transfected a rat fibroblast cell line F2408 with a recombinant EBNA2 expression plasmid and examined cell morphology, colony formation in soft agar, and tumorigenicity in nude mice. The morphology of transfected clones was similar to those of untransfected cells, but two of seven clones grew in soft agar, and four clones of seven clones reproducibly formed tumors in nude mice. These four clones showed EBNA2 expression, but non-tumorigenic clones did not. These results indicate that the expression of EBNA2 is correlated with tumorigenicity.

Animals↗

PubMed1994

Single amino acid substitution (58Pro-->Ser) in HTLV-I tax results in loss of ras cooperative focus formation in rat embryo fibroblasts.

Two tax genes cloned from a healthy HTLV-I carrier in whom the viral genome is clonally integrated into peripheral CD4+CD8+ cells showed a considerable difference in ras cooperative focus forming ability in rat embryo fibroblasts (REF). Sequence analysis revealed differences in two codons of the two genes. SH-1tax and SH-2tax. Studies using recombinants between these two tax genes showed that the deficiency in ability of SH-1tax to cooperate with ras in focus formation in REF was caused by a 58Pro-->Ser substitution. This amino acid substitution did not affect other tax functions such as colony formation in soft agar, focus formation in Rat-1 cells, immortalization of REF, and transcriptional activation through the CREB/ATF and NFkB/rel pathways. These results suggest that the domain of tax required for cooperative focus formation with ras in primary rat fibroblasts may be different from those required for other tax functions.

Animals↗

PubMed1994

Degradation of p53 only is not sufficient for the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts.

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein.

Base Sequence↗

PubMed1994

Regulation of early gene expression of human papillomavirus type 16 by inflammatory cytokines.

The host's immune reaction against human papillomavirus (HPV) infection remains poorly understood. Inflammatory cytokines undoubtedly play a key role through activating and coordinating the immune response. However, their direct interactions with the HPV genes remain unclear. In the present study, the effects of various inflammatory cytokines on HPV16 gene expression were investigated. In a CAT assay, tumor necrosis factor (TNF) alpha and interleukin-1 (IL-1) alpha were shown to repress HPV16 early gene expression at the transcriptional level through the noncoding region (NCR), whereas IL-6 and interferon-gamma did not. In Northern blot analysis, TNF and IL-1 were also shown to repress HPV16 E6/E7 mRNA expression in the HPV16-immortalized human keratinocyte cell line. The TNF- and IL-1-responsive elements in the HPV16 NCR were determined to lie within the cell-type-specific enhancer, where there are several binding sites for nuclear factors involved in HPV16 early gene regulation, suggesting the participation of these factors in TNF and IL-1 regulations. Thus, TNF and IL-1 were shown to have antiviral effects on HPV through down-regulation of its gene transcription. This is the first demonstration that TNF and IL-1 are involved in HPV gene regulation. These functions of inflammatory cytokines are presumed to contribute to the host's defense against HPV infection.

Cell Transformation, Viral↗

PubMed1994

Cervical/vaginal dysplasias of transgenic mice harbouring human papillomavirus type 16 E6-E7 genes.

Half of the female transgenic mice harbouring human papillomavirus type 16 (HPV-16) E6-E7 genes under control of the mouse mammary tumour virus promoter, developed malignant tumours, including salivary gland carcinomas, lymphomas and skin histiocytomas. Although the E6-E7 genes are aetiological factors for human anogenital carcinoma, the transgenic mice produced no tumours in the anogenital tract. We investigated cytological and histological changes in the anogenital tract of the same transgenic mice. Seventeen (77%) of 22 transgenic mice developed dysplastic and/or hyperplastic changes in the cervix and vagina. HPV-16 E6-E7 mRNA signals were observed in the genital lesions, while they were not detected in the normal cervices and vaginas of transgenic mice and control mice by RNA in situ hybridization analysis. RNA/PCR analysis using poly(A)+ RNA showed that only a full-length E6-E7 RNA was expressed in three scraped cell samples from dysplasia, whereas full-length and spliced E6-E7 transcripts were in three cell samples from dysplasia/hyperplasia. These results suggest that expression of both E6 and E7 genes of HPV-16 is important for inducing dysplastic and hyperplastic changes in the genital epithelium.

Animals↗

PubMed1994

Correlation between tumorigenicity and expression levels or splicing patterns of transcripts of the human papillomavirus type 16 E6 gene.

For determination of the correlation between tumorigenicity and the expression levels or splicing patterns of E6 mRNAs of the human papillomavirus type 16 in established cells, a vector containing the intact E6 open reading frame which expresses both unspliced and spliced mRNAs, one expressing only unspliced E6 mRNA, and one expressing both unspliced and spliced mRNAs but producing only truncated E6 proteins were constructed. In transformation assays and analyses of E6 mRNAs, a higher expression level of unspliced E6 mRNA was found to be closely associated with tumorigenicity. Furthermore, it was also related with anchorage-independent growth and a decreased serum requirement of the cells.

Alternative Splicing↗

PubMed1994

Involvement of human papillomavirus type 20 in epidermodysplasia verruciformis skin carcinogenesis.

The involvement of human papillomavirus (HPV) in skin carcinogenesis in a patient with epidermodysplasia verruciformis was studied. This patient had disseminated pityriasis versicolor-like lesions, flat warts, and a malignant skin carcinoma. HPV types 3, 17, 20 (HPV-20), and 38 were isolated and molecularly cloned from the benign skin lesions of this patient. Of these HPVs, only HPV-20 was detected in the malignant skin carcinoma. Transcripts of HPV-20 were also expressed in the carcinoma. These findings suggest that HPV-20 was involved in the skin carcinogenesis in this patient.

Adult↗

PubMed1993

NF-IL6 represses early gene expression of human papillomavirus type 16 through binding to the noncoding region.

The expression of human papillomavirus type 16 (HPV16) early genes, including E6 and E7 transforming genes, is regulated by several cellular factors binding to the noncoding region (NCR), such as the glucocorticoid receptor, NF-I, and AP1, all of which are positive regulators. We demonstrated that the nuclear factor for interleukin 6 expression (NF-IL6) specifically binds to the HPV16 NCR ranging from nucleotides 7007 to 7766 and represses the early gene expression of HPV16. The responsive element in HPV16 NCR was determined within the region ranging from nucleotides 7454 to 7766. In this region, many binding sites for other cellular transactivators, such as NF-I and AP1, have been detected. Interestingly, three of seven binding sites for NF-I and two of two binding sites for AP1 in this region overlap with the putative NF-IL6 binding sites identified by computer analysis. Competition experiments with the oligonucleotides containing such NF-I or AP1 sites indicated that NF-IL6 certainly binds to them. Furthermore, in a chloramphenicol acetyltransferase assay using mutant NF-IL6 expression vectors, the DNA binding domain of NF-IL6 was shown to be necessary for repression, whereas the functional domain was not. These findings indicate that repression may be caused by competition with other transcriptional activators, such as NF-I and AP1. Thus, NF-IL6 may play a significant role in the regulation of viral transcription as a part of the host's resistance to viral infection.

Acute-Phase Reaction↗

PubMed1993

A Ki-1-positive cell line expressing Epstein-Barr virus antigens, established from a child with Ki-1-positive lymphoma.

We report here the establishment of a CD30 (Ki-1) antigen-positive cell line 'Ki-JK' from a child with anaplastic large cell lymphoma. We characterized this cell line and show that: (i) Ki-JK cells do not grow in soft agar but infrequently produce a tumor or suppressed growth when injected into nude mice; (ii) Ki-JK cells expressed Epstein-Barr virus (EBV) antigens, EBV-associated nuclear antigen-2 and latent membrane protein; (iii) Ki-JK cells are labelled by in situ hybridization using an RNA probe derived from the BamHI W fragment of EBV, and (iv) polymerase chain reaction demonstrates the presence of an EBV BamHI W sequence in DNA of lymphoma and Ki-JK cells. These results suggest an etiological role for EBV in the development of some cases of Ki-1-positive lymphomas.

Adolescent↗

PubMed1993

Isolation of flat revertants from human papillomavirus type 18 E6E7 transformed 3Y1 cells by transfection with a rat embryo fibroblast cDNA expression library.

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes.

Animals↗

PubMed1992

Induction of uterine cervical neoplasias in mice by human papillomavirus type 16 E6/E7 genes.

We constructed a recombinant retrovirus containing the E6/E7 genes of human papillomavirus (HPV) 16 (ZE67) and examined the morphological changes in the cervicovaginal epithelium induced by inoculation of this virus into the vagina of mice. The ZNeo virus without HPV genes was used as a negative control. Moreover, cotreatment with phorbo-13-myristate-12-acetate or N-methyl-N'-nitro-N-nitrosoguanidine and these viruses was carried out. At the end of the observation period (9 months for CD-1 nu/nu and 15 months for C57BL/6J), of 31 CD-1 nude mice treated with ZE67, 7 and 19 had low-grade and high-grade dysplasia, respectively, while 5 of 22 mice treated with ZNeo had low-grade dysplasia (rank sum test, P less than 0.001). Similarly, 4 and 3 of 8 C57BL mice treated with ZE67 had low-grade and high-grade dysplasias, respectively, whereas 3 of the 8 control mice had low-grade dysplasias (P = 0.049). ZE67 plus phorbol-13-myristate-12-acetate and ZE67 plus N-methyl-N'-nitro-N-nitrosoguanidine cotreatments induced cervical cancers in 2 of 13 CD-1 nude mice and 6 of 15 C57BL mice, respectively. On the contrary, none of 6 CD-1 and 2 of 10 C57BL mice had cancer in the control groups (P = 0.0142 for phorbol-13-myristate-12-acetate treatment; P = 0.0173 for N-methyl-N'-nitro-N-nitrosoguanidine treatment). In addition, the existence and expression of HPV 16 E6/E7 genes were detected in the lesions induced by ZE67 but not in the lesions of the control mice by analysis by polymerase chain reaction and mRNA in situ hybridization. The present results suggest that HPV 16 E6/E7 genes induce dysplastic changes but require additional promoting or mutagenic stimulation for the development of cancer.

Animals↗

PubMed1992

Retrovirus gag protein p30 in the islets of non-obese diabetic mice: relevance for pathogenesis of diabetes mellitus.

We investigated the presence of retroviral protein in the pancreatic islets of non-obese diabetic mice to prove that the virus-like particle observed specifically in the pancreatic Beta cell of these mice was retrovirus. Western blot analysis probed with anti-retrovirus antibody demonstrated the existence of retroviral gag (group specific antigen) protein p30 in the islets of female non-obese diabetic mice. Islets of non-obese diabetic mice which were treated with cyclophosphamide, known to accelerate the development of insulitis and diabetes mellitus, have shown both a significantly increased number of retrovirus-like particles (type C) and enhanced expression of gag protein p30, compared to those of mice not treated with cyclophosphamide. These results confirmed the presence of type C retrovirus in non-obese diabetic mouse Beta cells and suggest a role for retrovirus in the development of insulitis and diabetes in these mice.

Animals↗

PubMed1992

Determination of the functional difference between human papillomavirus type 6 and 16 E7 proteins by their 30 N-terminal amino acid residues.

Human papillomavirus type 16 (HPV 16) is often found in cervical carcinomas, while HPV 6 is frequently associated with benign genital lesions. We have compared the abilities of the E7 transforming proteins of HPV 6 and 16 to transform various established and primary rodent cells by using the same heterologous promoter system. HPV 16 E7 efficiently induced anchorage-independent growth of all the rodent cell lines tested and immortalized or cooperated with ras in transforming primary rat cells. On the other hand, the transforming activity of HPV 6 E7 was lower and was restricted. By construction of chimeras of HPV 6 and 16 E7, we found that the difference in transforming activity between the two E7 proteins was mainly determined by the difference in their 30 N-terminal amino acid residues, although some activities seem to be slightly affected by differences in their residual C-terminal portions.

3T3 Cells↗

PubMed1992

Identification of antibodies against human papillomavirus type 16 E6 and E7 proteins in sera of patients with cervical neoplasias.

We have developed a sensitive and specific ELISA method using the s10-fusion proteins of human papillomavirus (HPV) 16 E6 and E7, expressed in E. coli. Sera from 97 women (30 patients with invasive cervical cancers, 26 patients with cervical intraepithelial neoplasia III (CIN III) and 38 healthy women) were tested for the presence of antibodies to E6 and E7 proteins. Eight (27%) of the 30 cervical cancer sera, five (19%) of the 26 CIN sera and none of the 38 normal sera were reactive with E6 proteins (cut-off point: absorbance (A) = 0.59, x +3SD). Ten (33%) of the 30 cervical cancer sera, two (8%) of the 26 CIN III sera and none of the 38 normal sera were reactive with E7 proteins (cut-off point: A = 0.40, x +3SD). The mean absorbance for anti-E7 antibody in positive cases was higher in cancer patients than in CIN III patients, while that for E6 did not differ between these two groups. Interestingly, six (50%) of 12 cancer sera which reacted with either E6 or E7 proteins were reactive for both proteins, whereas none of the sera from the CIN III patients reacted with both proteins. The high prevalence rates and high absorbance values for HPV 16 E6 and E7 antibodies in association with malignant transformation suggest that detection of these antibodies may be a useful diagnostic aid for cervical cancer-associated HPV 16.

Adult↗