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Biomedical subjects

A Hakura

Publications and source records attributed to A Hakura.

At least 55 records · Page 3Linked to original sources
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

Establishment and further characterization of a line of transgenic mice showing testicular tumorigenesis at 100% incidence.

We have reported production of transgenic mice containing human papillomavirus type 16 (HPV16) E6 and E7 oncogenes in which a characteristic testicular tumor develops at a very high incidence. Three transgenic mice transmitted the transgene to their siblings, in which the same type of tumor developed. In one line, named line 181, this testicular tumor developed in all the 93 males obtained for 10 generations. In most cases, this tumor was detectable bilaterally in the testes 9 to 10 months postdelivery. On cross-matings with other inbred strains, the HPV transgene was dominant in all the genetic backgrounds examined. In the condition of experimental cryptorchidism, obvious delay of tumor formation was observed. In these testes, the tumor cells were seen to arise from the interstitium. Moreover, this tumor also manifested obvious expression of gonadal specific 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and other enzymes for androgen metabolism. These observations strongly suggest that this tumor has originated from Leydig cells. This transgenic mouse line, therefore, provides a novel system for investigating in vivo carcinogenesis and the mechanism of transformation of male gonadal cells.

Androgens↗
PubMed1994

Detection of high-risk human papillomavirus in the cervix and semen of sex partners.

To investigate the possibility of sexual transmission of human papillomavirus (HPV), 53 married couples were examined for the presence of HPV-16 and -18 DNAs in the uterine cervix and semen using the polymerase chain reaction method. Twenty-three of the 53 women and 12 of the 53 male partners were positive for HPV-16 DNA. No HPV-18 DNA was detected in samples from any of the partners. In 27 pairs, both partners were negative for HPV DNA in cervix or semen; in the remaining 26 pairs, at least 1 of the partners was HPV-16-positive. In 9 (35%) of these 26 pairs, both partners were infected. Furthermore, 9 (75%) of the 12 women with HPV-positive partners were HPV-positive, while 9 (39%) of the 23 men with HPV-positive female partners were HPV-positive. These findings suggest an increased risk of HPV transmission via sexual intercourse, thereby underscoring the importance of preventive care against HPV infection during intercourse.

Adult↗
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

Detection and cloning of human papillomavirus DNA associated with recurrent respiratory papillomatosis in Thailand.

Recurrent respiratory papillomatosis (RRP) is highly prevalent in Thailand. In this study, we examined the presence of human papillomavirus (HPV) DNA in 25 RRP patients in Thailand by means of dot blot analysis and/or polymerase chain reaction. Eighty-four percent (21/25) of cases and 4% (1/25) of cases were positive for HPV-11 DNA and HPV-6 DNA, respectively. Three cases (3/25) were negative for all of the examined HPV types. No cases were positive for HPV-16 or 18. Furthermore, we isolated the recombinant HPV-11 DNA clone from a genomic library constructed with the DNA of RRP tissue. The restriction map of the cloned HPV DNA was identical with the map of known HPV-11 DNA. These results suggest at least that no specific HPV type or subtype is likely to be associated with RRP in Thailand.

Base Sequence↗
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

Suppression of viral transformation in rat embryo fibroblast cells immortalized with transforming genes of human papillomavirus type 16.

The E6 and E7 genes of human papillomavirus type 16 (HPV16) immortalize rat primary embryo fibroblast cells (REF) but do not transform them, irrespective of the expressions of the two genes. Previously, we reported that REF cells suppress the transformed phenotypes of a rat cell line transformed by the E6 and E7 genes of HPV16 in somatic hybrid cells. REF cells immortalized by HPV16 E6E7 genes (671 cells) also retained the ability to suppress E6E7-mediated transformation. Transformation of 671 cells by retroviruses containing middle T of polyomavirus, v-K-ras, or v-mos was also suppressed. 671 cells infected with retrovirus having middle T of polyomavirus (PyMLV) expressed sufficient middle T protein to transform a rat cell line. Cell fusion experiments showed that 671 cells suppressed transformation by middle T of polyomavirus without affecting the expression of middle T. These results suggest that REF cells immortalized by HPV16 E6E7 genes possess an intracellular function that suppresses transformation not only by E6E7 genes but also by other viral oncogenes including middle T of polyomavirus.

Animals↗
PubMed1993

Dimethyl sulfoxide (DMSO) is mutagenic for bacterial mutagenicity tester strains.

We used the Ames method with the modification of pre-incubation to evaluate the potential mutagenicity of DMSO. We performed the assays using nine different Ames Salmonella strains and Escherichia coli strains WP2 and WP2uvrA. DMSO was found to be mutagenic for Salmonella typhimurium TA1537 and TA2637 (the latter strain being isogenic to TA1537 but carrying plasmid pKM101) and for E. coli WP2uvrA. The mutagenic activity of DMSO observed at a concentration of 33% was about 10 times higher than the background level (65 revertants induced) for TA1537 after 20 min of incubation, where some lethal toxicity was observed. The mutagenicity of DMSO was observed in the presence and absence of rat liver S9 mix.

Animals↗
PubMed1993

New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535.

A new method for gene disruption in Salmonella typhimurium was developed. The key steps of this method are to produce restriction fragments with compatible ends, preligate to produce concatemers, and then transform by electrotransformation. We developed and used this method to construct a mutant of S. typhimurium TA1535 in which the resident ada-like (adaST) gene was replaced with a kanamycin resistance gene to produce an adaST-deletion mutant derivative. The S. typhimurium adaST-deletion strain did not exhibit a higher level of mutability upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine than did its wild-type parent strain. However, it did exhibit a higher sensitivity with respect to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The ability of AdaST to function as a transcriptional activator is discussed.

Bacterial Proteins↗
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↗