Biomedical subjects

T Shioda

Publications and source records attributed to T Shioda.

At least 19 recordsLinked to original sources

PubMed2006

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis.

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Animals↗

PubMed2001

Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein.

CITED1, a CBP/p300-binding nuclear protein that does not bind directly to DNA, is a transcriptional coregulator. Here, we show evidence that CITED1 functions as a selective coactivator for estrogen-dependent transcription. When transfected, CITED1 enhanced transcriptional activation by the ligand-binding/AF2 domain of both estrogen receptor-alpha (ERalpha) and ERbeta in an estrogen-dependent manner, but it affected transcriptional activities of other nuclear receptors only marginally. CITED1 bound directly to ERalpha in an estrogen-dependent manner through its transactivating domain, and this binding activity was separable from its p300-binding activity. CITED1 was strongly expressed in nulliparous mouse mammary epithelial cells and, when expressed in ER-positive MCF-7 breast cancer cells by transduction, exogenous CITED1 enhanced sensitivity of MCF-7 cells to estrogen, stabilizing the estrogen-dependent interaction between p300 and ERalpha. The estrogen-induced expression of the transforming growth factor-alpha (TGF-alpha) mRNA transcript was enhanced in the CITED1-expressing MCF-7 cells, whereas estrogen-induced expression of the mRNA transcripts for progesterone receptor or pS2 was not affected. Chromatin immunoprecipitation assay revealed that endogenous CITED1 is recruited to the chromosomal TGF-alpha promoter in MCF-7 cells in an estrogen-dependent manner but not to the pS2 promoter. These results suggest that CITED1 may play roles in regulation of estrogen sensitivity in a gene-specific manner.

Animals↗

PubMed2001

Over-expression of MSG1 transcriptional co-activator increases melanin in B16 melanoma cells: a possible role for MSG1 in melanogenesis.

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.

Animals↗

PubMed2001

Novel polymorphisms in human macrophage inflammatory protein-1 alpha (MIP-1alpha) gene.

Human macrophage inflammatory protein-1 alpha (MIP-1alpha) is a chemotactic cytokine, which binds to macrophages, T cells, and B cells affecting their activation. We found novel polymorphisms at four sites within MIP-1alpha gene in Japanese population: C to T in exon 2; A to G in intron 2; C to G and A to G in exon 3. They occurred on the same allele. Although MIP-1alpha effectively suppresses the replication of HIV-1 in vitro, we observed no statistically significant difference in the allele frequency of this polymorphism between HIV-1-infected and uninfected individuals in Japanese population. Since an increased transcription level of MIP-1alpha has been reported to be associated with inflammatory diseases such as atopic dermatitis, we also investigated the frequency of these polymorphisms among patients with atopic dermatitis, HIV-1-infected individuals (with a normal IgE level), and healthy donors. A small increase in ratio of homozygotes to other genotypes was observed in patients with atopic dermatitis (P = 0.04).

Alleles↗

PubMed2001

Sendai virus C protein physically associates with Stat1.

BACKGROUND: The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined. RESULTS: Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN-alpha/beta and IFN-gamma signalling. We found that both unphosphorylated Stat1 and tyrosine-phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low-salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1-HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY-Stat1 homodimer to pY-Stat1-HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high-salt buffer, and under this stringent (high-salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY-Stat1 were co-precipitated with anti-C antibody. CONCLUSION: The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.

DNA-Binding Proteins↗

PubMed2001

Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions.

The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed when pol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.

Blotting, Western↗

PubMed2001

Naturally occurring deletional mutation in the C-terminal cytoplasmic tail of CCR5 affects surface trafficking of CCR5.

CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(-) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221-222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(-) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4(+) T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(-) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.

Amino Acid Sequence↗

PubMed2001

Quintuple deglycosylation mutant of simian immunodeficiency virus SIVmac239 in rhesus macaques: robust primary replication, tightly contained chronic infection, and elicitation of potent immunity against the parental wild-type strain.

We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Animals↗

PubMed2001

Aberrant expression of MSG1 transcriptional activator in human malignant melanoma in vivo.

MSG1 was originally isolated as a candidate pigmentation-related gene. MSG1 mRNA transcripts were expressed strongly in cultured human and mouse normal epidermal melanocytes, and in highly pigmented mouse melanoma cells, while its expression was very weak in cultured non-pigmented human melanoma cells. Thus, MSG1 was initially proposed to be a melanocyte-specific gene, and its possible role in pigmentation has been speculated. It was found recently that the MSG1 protein interacts functionally with Smad4, which plays a pivotal role in signal transduction of transforming growth factor-beta. In this study, we analyzed MSG1 protein expression by immunohistochemistry using human tumor samples from nevus and malignant melanoma to reveal its role in pigmentation and melanoma development in vivo. A relatively strong but heterogeneous expression of MSG1 protein was seen in melanomas compared with weak expression in nevi. In nevi, MSG1 expression was mostly confined to the pigmented region, while it was expressed in both pigmented and non-pigmented regions in melanoma. Intracellularly, MSG1 protein was localized in the cytoplasm of nevus cells, but was seen in both nuclei and cytoplasm of melanoma cells. These results support a hypothesis that MSG1 plays a role in pigmentation. It is also suggested that MSG1 may be involved in malignant transformation of pigment cells. Alternatively, the aberrant expression of MSG1 in melanoma cells might be due to the abnormal environment, including aberrant cytokine or growth factor expression, associated with melanoma formation.

Apoptosis Regulatory Proteins↗

PubMed2000

X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation.

It has been suggested that DNA methylation plays a crucial role in genomic imprinting and X inactivation. Using DNA methyltransferase 1 (Dnmt1)-deficient mouse embryos carrying X-linked lacZ transgenes, we studied the effects of genomic demethylation on X inactivation. Based on the expression pattern of lacZ, the imprinted X inactivation in the visceral endoderm, a derivative of the extraembryonic lineage, was unaffected in Dnmt1 mutant embryos at the time other imprinted genes showed aberrant expression. Random X inactivation in the embryonic lineage of Dnmt1 mutant embryos, however, was unstable as a result of hypomethylation, causing reactivation of, at least, one lacZ transgene that had initially been repressed. Our results suggest that maintenance of imprinted X inactivation in the extraembryonic lineage can tolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X inactivation in the embryonic lineage.

Animals↗

PubMed2000

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening.

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein-Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein-Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Animals↗

PubMed2000

The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.

The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.

Amino Acid Sequence↗

PubMed2000

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

Amino Acid Sequence↗

PubMed2000

Antibodies to molluscum contagiosum virus in the general population and susceptible patients.

BACKGROUND: Since many attempts to cultivate molluscum contagiosum virus (MCV) in vitro have been unsuccessful, it is difficult to prepare a large quantity of antigens. To assess the seroprevalence of antibodies against MCV in 508 subjects with or without clinical MCV infection, a truncated recombinant protein from open-reading frame MC133L was synthesized using Sendai virus expression system and applied to enzyme-linked immunosorbent assay as an antigen. OBSERVATIONS: Antibodies to MCV were present in 7 (58%) of 12 patients with molluscum contagiosum, 7 (6%) of 108 healthy controls, 7 (9%) of 76 with atopic dermatitis, and 7 (18%) of 39 patients with systemic lupus erythematosus, although no clinical MCV infection was observed in the latter 3 groups. Of 7 human immunodeficiency virus (HIV)-positive patients with molluscum contagiosum, 1 (14%) was antibody positive, compared with 5 (2%) of 266 HIV-positive patients without molluscum contagiosum. Serum samples from patients with atopic dermatitis and systemic lupus erythematosus showed a higher reactivity (P<.001) than those from healthy controls, while serum samples from HIV-positive subjects showed a lower reactivity (P<. 001). CONCLUSION: The humoral immune response to MCV is usually confined to patients with molluscum contagiosum and may be affected by the immunological condition of the host.

Adolescent↗

PubMed2000

Effect of certain chemicals on the reproduction of medaka (Oryzias latipes).

In order to understand the effects of estrogenic chemicals on fish reproduction, we exposed male medaka (Oryzias latipes) to a natural estrogen [17 beta-estradiol (17 beta-E2)] and three estrogenic chemicals [bisphenol-A, nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP)]. After two weeks' exposure, one male medaka was kept together with two female medaka for spawning, and the number of eggs and hatchings were compared to those of a negative control group. The results indicated that exposure to 17 beta-E2 caused a significant decrease in the number of eggs and hatchings as compared to the negative control group at and above 3 nmol/l. Also, the highest concentrations of bisphenol-A and NP caused a decrease in the number of hatchings, but no decrease in hatchings was observed in DEHP treatments. In the treatment using these chemicals the decrease in egg numbers was not so much as in hatching numbers. When compared to other in vitro studies, concentrations observed to have adverse effects on reproduction in this study are generally lower. In addition, it was suggested that physical alterations, such as an induction of plasma vitellogenin, were caused at much lower concentrations than those at which a decline in reproductivity was actually induced.

Animals↗

PubMed2000

Isolation and expression of a novel member of the CITED family.

Chicken CITED3 (cCITED3) is a novel gene, which is expressed in the pre-somitic mesoderm, the mesonephric tubules, the Wolffian ducts and collecting tubules of the developing urogenital system and in the cranial sensory ganglia. Sequence analysis revealed that cCITED3 encodes a protein which contains two conserved domains that have been described for members of the CITED family.

Amino Acid Sequence↗

PubMed2000

A new polymorphism in the promoter region of the human interleukin-16 (IL-16) gene.

Interleukin 16 (IL-16) is a chemotactic cytokine which binds to CD4 and affects T cell activation. Here we report a novel single nucleotide polymorphism, T to C, in the promoter region of the IL-16 gene in two distinct Asian populations, Japanese and Thai. This mutation occurs at an allele frequency of approximately 22% and 18%, respectively. Although IL-16 potently suppresses replication of human immunodeficiency virus type 1 (HIV-1), we observed no significant difference in the allele frequency of this polymorphism between HIV-1-infected and non-HIV-1-infected individuals in both Asian populations. Since differential IL-16 levels have been reported to be associated with inflammatory diseases such as systemic lupus erythematosus, atopic dermatitis and allergic asthma, it would be of interest to analyze the allele frequency of this mutation in patients with these autoimmune and allergic diseases.

Gene Frequency↗

PubMed2000

Crystal structure of recombinant native SDF-1alpha with additional mutagenesis studies: an attempt at a more comprehensive interpretation of accumulated structure-activity relationship data.

Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.

Amino Acid Motifs↗