Takuya Okazaki

The University of Tokyo, Edo, Tōkyō, Japan

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Publications (3)18.23 Total impact

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    ABSTRACT: To elucidate the epigenetic regulation of Tat-independent human immunodeficiency virus (HIV) transcription following proviral integration, we constructed an HIV type 1 (HIV-1)-based replication-defective viral vector that expresses a reporter green fluorescent protein (GFP) product from its intact long terminal repeat (LTR). We transduced this construct into human tumor cell lines that were either deficient in or competent for the Brm-type SWI/SNF complex. One day after transduction, single cells that expressed GFP were sorted, and the GFP expression profiles originating from each of these clones were analyzed. Unlike clones of the SWI/SNF-competent cell line, which exhibited clear unimodal expression patterns in all cases, many clones originating from Brm-deficient cell lines either showed a broad-range distribution of GFP expression or were fully silenced. The resorting of GFP-negative populations of these isolated clones showed that GFP silencing is either reversible or irreversible depending upon the proviral integration sites. We further observed that even in these silenced clones, proviral gene transcription initiates to accumulate short transcripts of around 60 bases in length, but no elongation occurs. We found that this termination is caused by tightly closed nucleosome-1 (nuc-1) at the 5' LTR. Also, nuc-1 is remodeled by exogenous Brm in some integrants. From these results, we propose that Brm is required for the occasional transcriptional elongation of the HIV-1 provirus in the absence of Tat. Since the Brm-type SWI/SNF complex is expressed at marginal levels in resting CD4+ T cells and is drastically induced upon CD4+ T-cell activation, we speculate that it plays crucial roles in the early Tat-independent phase of HIV transcription in affected patients.
    Full-text · Article · Oct 2009 · Journal of Virology
  • Norihiko Okochi · Takuya Okazaki · Hideshi Hattori
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    ABSTRACT: We made micropatterned vascular endothelial cells, which have a regular capillary tube-like structure, on a bioactive hydrogel matrix. We applied a stamping method to transfer micropatterned bovine aortic endothelial cells to a growth factor-reduced basement membrane matrix (Matrigel) and type I collagen gel. In this study, we addressed the issues of how to accelerate cell transfer and the effective factors in doing so. We focused on the effects of the cell-substratum and cell-cell adhesiveness prior to applying cultured endothelial cells to a hydrogel matrix on cellular behavior under transfer printing. We found that individual cells cultured sparsely on substrata with different cell adhesivity transferred to Matrigel up to 40%, whereas cells cultured on patterned substrata having lines of 60 mum in width, which involved cell-cell contacts, could transfer homogeneously to Matrigel within a few hours. The morphology of such cells changed from a tape-like monolayer into a thinner, tube-like structure. The speed and the ratio of transfer of micropatterned cells to Matrigel were affected by the period of cell culture on micropatterned substrata. We also found that the intensity of vascular endothelial cadherin staining at cell-cell junctions of micropatterned cells was correlated with cellular behavior when applying them to Matrigel, on which cells formed a tube-like structure or to which cells migrated individually. Furthermore, micropatterned cells made regular tube-like structures when applied to type I collagen gel. Such tube-like endothelial cells had good viability. These findings may be useful for creating in vitro angiogenesis assays and tissue-like constructs that include capillary-like networks of vascular endothelial cells.
    No preview · Article · Jun 2009 · Langmuir
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    ABSTRACT: The mammalian SWI/SNF chromatin remodeling complex, an essential epigenetic regulator, contains either a single Brm or BRG1 molecule as its catalytic subunit. We observed frequent loss of Brm expression but not of BRG1 in human gastric cancer cell lines. Treatment with histone deacetylase inhibitor rescued Brm expression, indicating epigenetic regulation of this gene, and an RNA interference-based colony formation assay revealed antioncogenic properties of Brm. Brm immunostaining of 89 primary gastric cancers showed an obvious reduction in 60 cases (67%) and a severe decrease in 37 cases (42%). Loss of Brm is frequent in the major gastric cancer types (well- or moderately-differentiated tubular adenocarcinoma and poorly-differentiated adenocarcinoma) and positively correlates with the undifferentiated state. Among the minor gastric cancer types, Brm expression persists in signet-ring cell carcinoma and mucinous adenocarcinoma, but a marked decrease is observed in papillary adenocarcinoma. Intestinal metaplasia never shows decreased expression, indicating that Brm is a valid marker of gastric oncogenesis. In contrast, BRG1 is retained in most cases; a concomitant loss of BRG1 and Brm is rare in gastric cancer, contrary to other malignancies. We further show that Brm is required for villin expression, a definitive marker of intestinal metaplasia and differentiation. Via regulating such genes important for gut differentiation, Brm should play significant roles in determining the histologic features of gastric malignancy.
    Full-text · Article · Dec 2007 · Cancer Research