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Biomedical subjects

B Simizu

Publications and source records attributed to B Simizu.

At least 37 records · Page 2Linked to original sources
PubMed1994

Transcription-modulatory activity of full-length E6 and E6*I proteins of human papillomavirus type 16.

The E6 gene of human papillomavirus type 16 (HPV16) has the potential to encode full-length as well as truncated E6 proteins (E6*I and E6*II) by alternative splicings. Spliced ORF E6*I is considered to facilitate the translation of the neighboring E7 ORF; however, the putative E6*I protein is suspected to be functionless. In this study, the transcription-modulatory functions of full-length E6 and E6*I proteins were examined using cDNAs from a cervical carcinoma cell line. E6*I cDNA was able to trans-activate the autologous P97 promoter and the heterologous adenovirus E2 promoter. Full-length E6 was found to trans-activate the heterologous promoter, but repress transcription from the autologous P97 promoter. The transcription-modulatory functions of full-length E6 and E6*I proteins suggested that transcriptional regulation of HPVs associated with mucosal malignant lesions is complex.

Adenovirus E2 Proteins↗
PubMed1994

Detection of human papillomavirus DNA and state of p53 gene in Japanese penile cancer.

The frequency of integration with human papillomavirus (HPV) and its genotypes in Japanese penile cancer was examined with relation to p53 gene mutations using polymerase chain reaction amplification. Tissues were obtained from 13 patients (eight from freshly frozen and five from paraffin-embedded samples). HPV DNA was detected in seven out of the 13 (54%), and their genotypes were type 16 in four, type 31 in one and type 33 in two cases. Neither HPV-detected nor -undetected tissues showed mutated alterations in exons 4-9 of p53 genes. The results suggest HPV to be, at least to some extent, involved in the oncogenesis of penile cancer, and that p53 gene mutations may not correlate with the development of penile cancer.

Aged↗
PubMed1993

Translational properties of the human papillomavirus type-6 L1-coding mRNA.

A cDNA encoding a bicistronic mRNA, E1E4L1, which was generated by double-splicing of the E1, E4 and L1 genes, of the type-6 human papillomavirus (HPV-6), was cloned. The E1E4 and L1 open reading frames (ORFs) in this cDNA were expressed in COS-1 or CV-1 cells as fusion proteins with Escherichia coli beta-galactosidase (beta Gal), and the products were analyzed by immunoprecipitation and enzyme assay. The results showed that the translational efficiency of the L1 ORF was about 9-15-fold less efficient than that of the E1E4 ORF. Substitution of the ATG of the E1E4 ORF with AAG increased translation of the L1 ORF about 30-fold. Lengthening of the intercistronic sequence to 31 bp, equivalent in length to the bicistronic HPV-1 mRNA, showed little translational effect relative to the wild-type 12-bp intercistronic sequence. (Carets [] represent splicing of RNA.)

Animals↗
PubMed1993

Expression of alternatively spliced src messenger RNAs related to neuronal differentiation in human neuroblastomas.

Neuroblastoma, the most common malignant solid cancer of children, has an ability to differentiate in vitro and in vivo. This biological property has a significant influence upon the prognosis of patients with neuroblastomas. Neuronal cells express three alternatively spliced forms of c-src mRNA (nonneuronal c-src, neuronal c-srcN1, and neuronal c-srcN2), which are found at different levels in adult and fetal human brain tissue. In this study, the transcriptional levels of the three c-src mRNAs were examined in relation to the neural differentiation in eight human neuroblastoma cell lines and two clonal sublines and in seven primary neuroblastoma tissues by S1 nuclease protection assays. Neuronal c-srcN1 mRNA was expressed at high levels in neuroblastoma cell lines with the ability to differentiate but not in the cell lines lacking the capacity to mature in response to chemical inducers irrespective of N-myc gene amplification and overexpression. In terminally differentiated neuroblastoma cells, the expression of neuronal c-srcN2 mRNA, which was barely detectable at a steady-state level in the uninduced cells, increased to significant levels. Infantile neuroblastomas identified by mass screening tests expressed both neuronal c-srcN1 and c-srcN2 mRNAs at levels almost identical to that found in human brain tissue, but terminally differentiated neuroblastoma cells, neuroblastomas from older children identified based on clinical symptoms, did not. These results suggest that neuronal c-src expression and the ability of neuroblastomas to differentiate in vitro and in vivo may be correlated.

Base Sequence↗
PubMed1993

Paratesticular neuroblastoma with N-myc activation.

The authors describe a case of disseminated neuroblastoma discovered as a paratesticular tumor in a 7-month-old boy. The ectopic adrenal tissues adjacent to the paratesticular tumor and multiple lesions in the adrenal gland and skin suggested the possibility of multifocal primary tumors. Although infantile neuroblastoma diagnosed at less than 1 year of age generally responds well to treatment irrespective of distant metastases, metastases developed, and the boy died of disease within 7 months. All multiple lesions had amplification and overexpression of the N-myc protooncogene, which might explain the aggressive phenotype of this rare case.

Blotting, Northern↗
PubMed1992

Amplification and overexpression of the c-erbB-2 protooncogene in human gastric cancer.

The c-erbB-2 protooncogene encodes a possible growth factor receptor. This gene has been studied as to whether it can be regarded as a prognostic indicator in human breast carcinoma. As amplification and overexpression of the gene have been reported in several adenocarcinomas, 24 specimens of human gastric cancers were examined by immunohistochemical staining (24 cases), by Southern blotting (23/24) and by Northern blotting (16/24). Amplification of the gene was detected in two moderately differentiated tubular adenocarcinomas (8.7%), and overexpression of c-erbB-2 mRNA was detected in three moderately differentiated tubular adenocarcinomas (18.8%). By immunohistochemical staining of paraffin-embedded tissues using a polyclonal antibody to c-erbB-2 gene products, the cell membrane was stained positively in three cases of gastric cancers which overexpressed c-erbB-2 mRNA. Peritoneal metastases were found in six gastric cancers, including two moderately differentiated tubular adenocarcinomas in which amplification of c-erbB-2 occurred. These results suggest that amplification and overexpression of c-erbB-2 may be correlated with metastases in differentiated adenocarcinoma of the stomach.

Adenocarcinoma↗
PubMed1992

Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.

Androgens↗
PubMed1992

The induction of IL-6 and gelatinase B by IL-1 in mouse cell lines transformed with bovine papillomavirus: decreased production in tumorigenic cells.

Six cell lines, that were cloned from murine C127 cells infected by bovine papillomavirus type 1 (BPV1), were found to differ in the degree of transformation in vitro and of tumorigenicity in vivo. In these cell lines the degree of tumorigenicity was inversely correlated with IL-6 induction by IL-1 beta. Whereas the parental C127 cell line produced 15-30 U/ml of IL-6 spontaneously, none of the transformed cell lines produced significant levels of IL-6 constitutively. On induction by human IL-1 beta the parental C127 cell line produced up to 300 U/ml of IL-6, whereas the fully transformed ID14 cell line failed to produce any. The less transformed cell lines produced lower yields of IL-1 beta-induced IL-6, dependent on their degrees of transformation and tumorigenicity. Gelatinase B (96 kDa), a matrix metalloproteinase inducible by IL-1 beta, was dose-dependently regulated in the parental C127 cell line and in the weakly transformed cell line Tlc. These data suggest that transformation processes by BPV1 generally impair IL-1-regulated gene transcription. This impairment seems not to be located at the IL-1 beta receptor level, since in all the cell lines studied the numbers and affinities of the IL-1 beta binding sites were found to be comparable. This impairment seems not to be mediated by transformation-induced inactivation of the protein kinase C pathway since phorbol 12-myristate 13-acetate (PMA) induced IL-6 production equally well in all C127 cell-derived clones. It is suggested that BPV1 transformation can change the expression of host genes that might play a functional role in tumor immune surveillance and tumorigenicity in vivo.

Animals↗
PubMed1992

[Evolution and ecological changes of animal viruses].

Although the origin of viruses has not yet been clarified, definite differences in evolutionary patterns have been found among RNA. Retro and DNA viruses. These differences are reflected in infectious diseases. RNA viruses, which have RNA in their genome, replicate many times over in cells within a short period of time, destroying the host cells and causing an acute infection. As the error frequency of RNA replicase enzymes is high, the rate of evolution of RNA viruses is very rapid. Retroviruses also contain RNA as their genome, but the genome RNA is reversely transcribed to the DNA in nuclei and then incorporated into the host chromosome to replicate. The error frequency of reverse transcriptase is also high, and therefore mutations easily occur as well. The transcribed DNA is integrated into host DNA in the nucleus, and it remains in the integrated state for human entire life time, causing chronic disease or developing malignant tumors. As DNA viruses except poxviruses replicate inside the cell nucleus and the error frequency of DNA polymerase is low, the speed of mutation and the degree of resulting diversity are lower than those in the case of the RNA virus. DNA viruses tend to stay inside the body for long periods of time and easily become latent. In this paper, I shall discuss 1) the nature of viruses, 2) the origin of viruses, 3) mutation and recombination, 4) diversity of RNA viruses, 5) quickly changing viral diseases, 6) eradicated viral diseases, 7) chronic and malignant diseases, and 8) control of viral diseases.

Biological Evolution↗
PubMed1991

Expression of N-myc and c-src protooncogenes correlating to the undifferentiated phenotype and prognosis of primary neuroblastomas.

Genomic amplification of the N-myc protooncogene in neuroblastomas correctly predicts poor outcome for the patients. However, the prognosis for neuroblastomas with a single copy of N-myc is also poor in cases diagnosed after 1 year of age but good in infantile cases. To elucidate the different prognoses depending upon the age of the patients with neuroblastoma, we performed an analysis of the expression of protooncogenes related to neural differentiation. We examined the genomic amplification of N-myc in 26 specimens of neuroblastomas and further analyzed 22 of the 26 cases for expression of N-myc, c-src, c-Ha-ras, and c-fos. Consequently, we observed frequent overexpression of N-myc in undifferentiated neuroblastomas and enhanced expression of c-src and c-Ha-ras in infantile neuroblastomas with favorable prognosis and in neuroblastomas differentiated by chemotherapy. These findings suggest that c-src and c-Ha-ras play important roles in the neural differentiation of infantile neuroblastomas.

Blotting, Northern↗
PubMed1991

Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis.

The presence of human papillomavirus (HPV) DNA in oral, sinus, pharynx, and larynx lesions of Japanese patients was studied by Southern blot hybridization under less stringent (25% formamide, 42 degrees C) and stringent (50% formamide, 42 degrees C) conditions. Three samples from 10 benign tumors, and 3 of 30 malignant tumors, contained HPV DNA or HPV-related sequences. The HPV DNAs harbored in three laryngeal papillomas were HPV-11, -6, and -6 or -11, respectively. The HPV DNA and viral capsid antigens were easily detected by in situ hybridization, Western blotting, and peroxidase-antiperoxidase staining. However, neither the typical restriction pattern of HPV DNA nor viral antigen was identified in the malignant tumors, suggesting that subgenomic fragments remained integrated in the host cell DNA.

Antigens, Viral↗
PubMed1991

Quantitative detection of spliced E6-E7 transcripts of human papillomavirus type 16 in cervical premalignant lesions.

The splicing patterns of E6-E7 transcripts of human papillomavirus type 16 (HPV16) in cervical premalignant lesions were quantitatively analyzed by S1 nuclease protection assay. The major E6-E7 transcripts in HPV16-containing cervical lesions (four cervical intraepithelial neoplasias and one invasive carcinoma) were from spliced E6*I/E7 mRNA. The unspliced E6/E7 mRNA, which can encode the full-length zinc finger protein E6, is expressed as 8 to 15% of E6-E7 transcripts. The spliced E6*II/E7 mRNAs were expressed as 14 to 24% of E6-E7 transcripts in most tissues. However, in HPV 16-containing cell lines, the expression levels of spliced and unspliced E6-E7 transcripts were variable.

Blotting, Southern↗
PubMed1991

Human papillomavirus type 6 and 11 E4 gene products in condyloma acuminata.

The human papillomavirus type 6 (HPV-6) E4 gene was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (E4-beta-Gal), and rabbit antibody against the E4-beta-Gal was prepared. By Western blotting with this antibody, we detected E4 gene products in six out of 18 condyloma acuminata specimens. In four specimens (C-1, C-13, C-14 and C-19), the E4 protein was found as a 10K/11K doublet, but in other specimens (C-8 and C-23), only the 11K protein was detected. By Southern blot analysis, it was found that C-13 harboured HPV-6 DNA but that C-1 and C-8 harboured HPV-11 DNA, indicating that the E4 proteins of HPV-6 and -11 have cross-reactive antigenicity. After incubation at 37 degrees C of the C-23 tissue specimen, the 10K protein was clearly detected. These results suggest that the 10K protein may be derived from the 11K protein by a modification such as proteolytic cleavage before and/or after specimens were taken.

Base Sequence↗
PubMed1989

Detection of the human papillomavirus 6b E2 gene product in genital condyloma and laryngeal papilloma tissues.

We have previously reported that the E2 open reading frame of human papillomavirus type 6b (HPV6b) was expressed in Bombyx mori cell line Bm-N as a fusion protein with baculovirus polyhedrin. Here we show that antibody against this E2-polyhedrin fusion protein recognized a putative gene product as a 49-kDa protein in some genital condyloma and laryngeal papilloma tissues, which were associated with HPV6 and HPV11, by immunoblotting analysis. The antibody reacted also with antigens in the nucleus of cells in the middle and upper layers of these papilloma tissues.

Animals↗
PubMed1989

Characterization of bovine papillomavirus type 1-transformed clones which show distinct transformed phenotypes.

Seventeen independent cell clones were isolated from C127 cells transformed by bovine papillomavirus type 1 (BPV-1). Transformants showed differing degrees of expression of the transformed phenotype as monitored by saturation density, doubling time, growth in medium with a low serum concentration and colony-forming efficiency in soft agar. The degree of expression of the transformed phenotype did not correlate with either the BPV-1 copy number or levels of BPV-1-specific RNA in the transformed cell clones. A characteristic transformed cell clone, T1c, showed the lowest degree of expression of the transformed phenotype but contained the highest copy number of BPV-1 DNA and the highest level of BPV-1-specific mRNA. When we analysed different transformants by two-dimensional gel electrophoresis, we found that a set of six proteins changed quantitatively. Changes in the expression of these proteins were most consistent in clones expressing the greatest number of parameters of transformation, e.g. clone T4a. These data indicate that changes in the expression of cellular genes may correlate with the degree of expression of the transformed phenotype.

Animals↗
PubMed1988

Expression of human papillomavirus type 6b E2 gene product with DNA-binding activity in insect (Bombyx mori) cells using a baculovirus expression vector.

Human papillomavirus type 6b (HPV6b) has been shown to be a major etiologic agent of genital condylomas. The E2 gene, one of the early genes, has been shown to activate the enhancer element in trans in cells transformed with bovine papillomavirus type 1a (BPV1a) but the E2 gene product of any HPV has not been identified. The E2 gene of HPV6b was inserted into the polyhedrin gene of a Baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), 156 bp downstream from the translational start codon, and transferred into BmNPV by allelic replacement in a cotransfected Bombyx mori cell line, Bm-N. The predicted 46-kDa protein was produced by a recombinant virus in the infected Bm-N cells at a high level under the control of the polyhedrin promoter. We obtained the antibody against the putative E2-polyhedrin fusion protein by immunizing a rabbit with this protein. This protein reacted with the antibodies against polyhedrin and the fusion protein. This protein did specifically bind to the upstream regulatory region of the HPV6b and BPV1a genomes. This DNA binding activity was blocked by the antibody against this protein.

Animals↗
PubMed1988

Expression of the E2 open reading frame of papillomaviruses BPV1 and HPV6b in silkworm by a baculovirus vector.

To produce and characterize the nature of the E2 protein of papillomaviruses, we have developed a system to express the DNA sequence containing an open reading frame (ORF) as a fusion protein in cultured insect cells and silkworms. The DNA fragments of the E2 ORF predicted from the DNA sequence of bovine papillomavirus type 1 and human papillomavirus type 6b were linked to the N-terminal part of polyhedrin gene of the Baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) vector. Hybrid proteins composed of polyhedrin protein (52 amino acids) and the E2 proteins of BPV1 (410 amino acids) or HPV6b (346 amino acids) were efficiently produced in B. mori cells and silkworm larvae infected with recombinant viruses. The amount of E2 fusion proteins produced by recombinant viruses was comparable to that of polyhedrin produced by wild type BmNPV. The hybrid proteins were immunologically reactive to antiserum against polyhedrin.

Animals↗