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Biomedical subjects

B Simizu

Publications and source records attributed to B Simizu.

At least 55 records · Page 3Linked to original sources
PubMed1988

Transcriptional differences of the human papillomavirus type 16 genome between precancerous lesions and invasive carcinomas.

Human papillomavirus type 16 (HPV16) genome DNA and its transcripts in biopsied cervical neoplasias were analyzed by simultaneous extraction of DNA and RNA from one biopsied sample. Southern blot analysis revealed that 5 of 20 cervical intraepithelial neoplasias (CINs) contained HPV16 DNAs existing primarily as episomes and two of seven invasive carcinomas harbored HPV16 genome sequences integrated into the host DNA. Northern (RNA) blot analysis showed that the HPV16 genome sequences were transcriptionally active in the five CINs, as well as in the two invasive carcinomas. The pattern of HPV16-specific transcripts in the CINs was uniform, and the major transcripts were 4.2, 2.2, 1.6, and 1.4 kilobases in size. However, the pattern of HPV16-specific transcripts in the invasive carcinomas was variable and different from that in CINs, suggesting that the alteration of transcriptional pattern might play a key role in the development of malignancy.

Carcinoma↗
PubMed1987

Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-beta-galactosidase fusion proteins and their antigenic properties.

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.

Antibodies, Viral↗
PubMed1987

Integration and transcription of human papillomavirus type 16 and 18 sequences in cell lines derived from cervical carcinomas.

Five cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E1 ORF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the L1 and L2 ORFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.

Base Sequence↗
PubMed1987

Expression of human papillomavirus types 6b and 16 L1 open reading frames in Escherichia coli: detection of a 56,000-dalton polypeptide containing genus-specific (common) antigens.

The human papillomavirus (HPV) genome contains two large open reading frames (ORFs), designated L1 and L2. To characterize the antigenic properties of the L1 ORF-encoded proteins, we cloned the L1 ORFs of HPV6b and HPV16 in plasmids, and these were expressed in Escherichia coli. First, the HPV6b DNA, representing 85.2% of the L1 ORF, was cloned in pUC19 and expressed in E. coli JM83 and RB791 as a 160,000-molecular-weight (160K) fusion protein with E. coli beta-galactosidase (6bL1/beta-gal). Second, the HPV16 DNA, representing 89.8% of the L1 ORF, was cloned in pKK233-2 and expressed as a 56K protein (16L1) in strain RB791. Both the 6bL1/beta-gal and 16L1 proteins cross-reacted with anti-bovine papillomavirus type 1 (BPV1) antibody raised against disrupted BPV1 particles. An antibody raised against the 6bL1/beta-gal fusion protein reacted with the 16L1 protein and also with native papillomavirus antigens in human genital condyloma and bovine fibropapilloma tissues, as determined by biotin-streptavidin staining. Furthermore, the anti-6bL1/beta-gal antibody recognized a 54K protein which seemed to be a major capsid protein of BPV1 and also a 56K protein of biopsies harboring HPV6 or HPV11. From these results we concluded that the papillomavirus L1 gene product contains genus-specific (common) antigens and that the HPV6 and HPV11 L1 genes specify the 56K capsid protein.

Animals↗
PubMed1987

The c-sis gene expression in cells from a patient with acute megakaryoblastic leukemia and Down's syndrome.

The c-sis gene expression in leukemia cells from a patient with acute megakaryoblastic leukemia and Down's syndrome was studied. The leukemia blasts were identified as megakaryoblasts by the platelet peroxidase reaction and the reactivity against antiplatelet monoclonal antibodies. Leukemia cells obtained from peripheral blood or bone marrow specimens before and after initiation of chemotherapy were analyzed for c-sis gene expression by the RNA-DNA dot blot hybridization. Although the level varied, the mRNA of the c-sis gene was detected in all megakaryoblastic leukemia cells obtained at different clinical stages. The c-sis mRNA level in cells obtained after relapse was higher than that obtained before the initiation of therapy. The 25S c-sis mRNA was detected in a megakaryoblastic leukemia cell line established from this patient. The role of the expression of the c-sis gene in acute megakaryoblastic leukemia cells is discussed.

Acute Disease↗
PubMed1986

Detection of human papillomavirus type 16 DNA and evidence for integration into the cell DNA in cervical dysplasia.

The presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23% of mild dysplasias, 32% of moderate dysplasias, 55% of severe dysplasias and 62% of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 86% of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.

Carcinoma in Situ↗
PubMed1986

Detection of human papillomavirus DNA in genital warts, cervical dysplasias and neoplasias.

Biopsies from human genital lesions in Japan (108 samples), including condyloma acuminata, squamous metaplasia, dysplasia, and cervical cancer, were screened for the presence of human papillomavirus (HPV) 6-, 11-, 16-, and 18-related DNAs by spot hybridization and Southern blot hybridization under stringent conditions. By spot hybridization, HPV 6/11-related DNA was found in 92.9% (13/14) of condyloma acuminata and in 6.7% (1/15) of cervical cancer biopsies. HPV 16/18-related DNA was found in 37.7% (5/14) of cervical cancer biopsies and was exclusively associated with invasive squamous cell cancer, 66.7% (8/12), and with cervical carcinoma in situ, 50% (9/18). Some sample DNAs were further characterized by restriction enzyme digestion followed by Southern blot analysis, and we confirmed the presence of HPV-specific DNA fragments and mixed infection with both HPV 6/11- and HPV 16/18-related HPVs in one lesion.

Adult↗
PubMed1985

Properties of monoclonal antibodies against glycoproteins of western equine encephalitis virus.

To analyze the biological activities of the alphavirus glycoproteins, eight different monoclonal antibodies against the two glycoproteins of western equine encephalitis virus were isolated. Five of the eight monoclonal antibodies were shown to be specific for E1 and three for E2 protein by an enzyme-linked immunosorbent assay and by radioimmunoprecipitation. Three of the five anti-E1 and all of the anti-E2 monoclonal antibodies inhibited hemagglutination by purified virions. One anti-E1 and two anti-E2 monoclonal antibodies possessed high virus-neutralizing activity.

Antibodies, Monoclonal↗
PubMed1984

Structural proteins of Chikungunya virus.

Polyacrylamide gel analysis of the structural proteins of African and Asian strains of Chikungunya virus, an alphavirus, showed that both strains contain three structural proteins: glycosylated E1 and E2, embedded in the viral envelope, and a nonglycosylated nucleocapsid protein. In pulse-chase experiments the precursor protein PE2 was chased into glycoprotein E2, which migrated slightly faster than did glycoprotein E1. The third Chikungunya glycoprotein, E3, was not associated with mature virions but was released into culture fluids. With glycoproteins E1 and E2, separated by glass wool column chromatography, it was shown that hemagglutinating activity is associated with glycoprotein E1.

Chikungunya virus↗
PubMed1983

A variant of western equine encephalitis virus with nonglycosylated E3 protein.

A variant clone (At/A125) of western equine encephalitis virus was isolated from a line of mosquito cells persistently infected with a temperature-sensitive parent strain, A125. Variant-infected cells produced an altered form of PE2 protein which migrated with a higher electrophoretic mobility than wild type or A125 PE2. The altered PE2, like PE2 of wild type, was precipitated by anti-envelope proteins serum but not by anti-E1 serum. In pulse-chase experiments the altered PE2 protein was shown to yield E2 of normal electrophoretic mobility in SDS-polyacrylamide gel electrophoresis. The unglycosylated form of the altered PE2 synthesized in the presence of tunicamycin migrated at the same position as the unglycosylated PE2 obtained from tunicamycin-treated, parent strain-infected cells. This suggested that migration difference might be ascribable to incomplete glycosylation of PE2, possibly of its E3 component. E3 is released into culture fluid of wild-type-infected cells as an approximately 11-kd glycoprotein, while variant-infected culture fluid yielded a smaller, apparently virus-specific protein. The protein could not be labeled with [3H]mannose, suggesting that the polypeptide moiety of E3 in the variant infected cells failed to be glycosylated. The parent strain, A125, and a revertant of the variant, At/A125/rev, did not synthesize such altered PE2 and E3 proteins. The growth of At/A125 in mosquito cells was similar to that of parent or wild type but depressed in vertebrate cells.

Aedes↗
PubMed1983

Mutants of sindbis virus. IV. Heterotypic complementation and phenotypic mixing between temperature-sensitive mutants and wild-type Sindbis and Western equine encephalitis viruses.

Heterotypic complementation between temperature-sensitive (ts) mutants of Sindbis (SIN) and Western equine encephalitis (WEE) viruses occurs under appropriate conditions. One heterotypic pair, SIN ts153 X WEE ts39 showed efficient complementation, and four other combinations gave detectable complementation, indicating that these two viruses, which are closely related serologically and biochemically, are sufficiently closely related to complement each other functionally. Cells mixedly infected with ts mutants or wild-type strains of both SIN and WEE viruses produced phenotypically mixed virions, in addition to both parental viruses. Various types of phenotypically mixed virions have been identified by neutralization with corresponding antisera, by thermal inactivation and by temperature sensitivity of replication. Some virions contained WEE genomes and envelopes containing primarily SIN proteins. Other phenotypically mixed virus yields contained primarily doubly neutralizable viruses which are presumed to have a mosaic of envelope proteins. Phenotypically mixed virions were morphologically indistinguishable from the parental types.

Encephalitis Virus, Western Equine↗
PubMed1983

Structural proteins of Western equine encephalitis virus: amino acid compositions and N-terminal sequences.

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.

Amino Acid Sequence↗