Biomedical subjects

R Hirochika

Publications and source records attributed to R Hirochika.

5 recordsLinked to original sources

PubMed1992

Thy-1 inhibits mitogen-induced Ca2+ oscillation in ras-transformed mouse fibroblasts.

Cell surface glycoprotein Thy-1 functions as a transformation suppressor in v-ras-transformed NIH/3T3 cells [Sugimoto et al., (1991) Cancer Res. 51, 99-104.]. In order to understand the mechanism of action of Thy-1, we examined the effect of Thy-1 expression on mitogen-induced Ca2+ oscillation which was correlated with v-ras-transformation [Fu et al., (1991) FEBS Lett. 281, 263-266.]. Forced expression of Thy-1 in v-ras-transformed cells inhibited mitogen-induced Ca2+ oscillation. Although v-Ras-free, Thy-1-positive NIH/3T3 cells (major population) did not show Ca2+ oscillation, whereas in Thy-1-negative NIH/3T3 cells (less than 1% of the population) Ca2+ oscillation was observed. Finally, replacement of the carboxyl-half of Thy-1 with that of CD4 abolished the inhibitory effect of Thy-1. These results suggest that Thy-1 directly or indirectly participates in the negative regulation of Ca2+ response by inhibiting Ca2+ oscillation.

3T3 Cells↗

PubMed1992

Somatic gene therapy for diabetes with an immunological safety system for complete removal of transplanted cells.

To develop somatic gene therapy for diabetes, we studied an animal model with proinsulin-producing fibroblasts with an immunological safety system. Cultured mouse fibroblasts of the Ltk- cell line were transfected first with the efficient human proinsulin expression vector pBMG-Neo-Ins. Initially, 2 x 10(6) cells with a proinsulin-production rate of 91 ng.24 h-1.10(6) cells-1 were transplanted i.p. into streptozocin-induced diabetic C3H mice. The blood glucose concentrations improved between the first and the 28th day, but the animals died of hypoglycemia between the 29th and 46th days. The proinsulin-producing Ltk- cells were further transfected with a second plasmid, pHEBo-CD8.2, encoding BALB/c mouse T-cell differentiation antigen. The CD8.2 allotype is different from CD8.1 allotype by only one amino acid substitution and should be only slightly antigenic to the recipient C3H mice. Somatic gene therapy with these doubly transfected cells followed by the consecutive administration of a monoclonal antibody to CD8.2 resulted in an initial decrease of blood glucose concentrations followed by the permanent recurrence of hyperglycemia, thus proving the complete removal of the transplanted cells. Cultured fibroblasts were thus proven capable of supplying sufficient proinsulin to lower the blood glucose concentrations in diabetic animals. The immunological safety system with a combination of artificial expression of cell surface antigen and the administration of the specific monoclonal antibody was an effective safety system for somatic gene therapy.

Animals↗

PubMed1988

Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins.

The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.

Chromosome Deletion↗

PubMed1988

Regulation of human papillomavirus type 11 enhancer and E6 promoter by activating and repressing proteins from the E2 open reading frame: functional and biochemical studies.

E2-C, a protein consisting mainly of the carboxy-terminal 45% of the human papillomavirus type 11 (HPV-11) E2 protein, was expressed from the Rous sarcoma virus long terminal repeat in mammalian cells. It competitively repressed the stimulatory action of the full-length E2 protein on the HPV-11 enhancer located in the upstream regulatory region, as assayed by the expression of a reporter gene from the simian virus 40 (SV40) early promoter in transiently transfected monkey CV-1 cells. A mutation in the initiation codon for E2-C protein eliminated repression. In the human cervical carcinoma cell line C-33A, which apparently lacks endogenous HPV DNA, the HPV-11 enhancer-SV40 promoter and the HPV-11 enhancer in its normal association with the E6 promoter had high constitutive activity. In these cells, E2 proteins had little or no stimulatory effect on the transcriptional activity of the HPV-11 enhancer-SV40 promoter. In contrast, the HPV-11 enhancer-E6 promoter was stimulated by the HPV-11 E2 protein but repressed by the bovine papillomavirus type 1 E2 protein, an effect due either to a quantitative difference in E2 expression levels or to a qualitative difference in the trans-activating abilities of the two E2 proteins. In this cell line, the HPV-11 E2-C protein suppressed both the constitutive activity and the HPV-11 E2 trans activation. The E2-C protein was also produced from an expression vector in Escherichia coli. The E2-C protein present in crude E. coli lysates or purified by DNA affinity chromatography associated in vitro with a specific sequence, ACCN6GGT, in filter-binding assays. Moreover, the protein generated DNase I footprints spanning this motif identical to those of bacterially expressed full-length E2 proteins. This DNA sequence motif is necessary and sufficient for E2 binding in vitro and enhancer trans activation in vivo (H. Hirochika, R. Hirochika, T. R. Broker, and L. T. Chow, Genes Dev. 2:54-67, 1988). Mutations in this sequence that abolished interactions with E2 also precluded binding to the E2-C protein. These data strongly suggest that the full-length E2 protein consists of two functional domains: the amino-terminal half for trans activation and the carboxy-terminal half for DNA binding. The mechanism by which E2-C represses E2-dependent enhancer activity most likely involves competition with E2 for binding to a common transcriptional regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning, Molecular↗

PubMed1987

A human papilloma virus type 11 transcript encoding an E1--E4 protein.

The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown.

Base Sequence↗