• Title/Summary/Keyword: cDNA representational difference analysis

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Porphyromonas Gingivalis Lipopolysaccharide Increases Monocyte Adhesion to Microvascular Endothelium by Induction of Adhesion Molecules

  • Kim, Su-Ryun;Park, Hyun-Joo;Bae, Soo-Kyung;Park, Ji-Hyun;Kim, Hyo-Sun;Koo, Tae-Hyeon;Bae, Moon-Kyoung
    • International Journal of Oral Biology
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    • v.33 no.4
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    • pp.149-154
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    • 2008
  • Porphyromonas gingivalis, a major periodontal pathogen, has been implicated in the initiation and progression of periodontal disease. Endothelial dysfunction (Editor note: Aberrant and dysfunction are somewhat redundant. The authors may want to choose one or the other.) contributes to chronic periodontal inflammation. Using cDNA-representational difference analysis, we found that P.gingivalis lipopolysaccharide differentially induces a number of genes in human microvascular endothelial cells. Among these upregulated genes, we focused on intercellular adhesion molecule-1 (VCAM-1), which is crucial for leukocyte recruitment during vascular inflammation. P. gingivalis LPS significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) as well as ICAM-1. Promoter assays revealed that the transcription of these cell adhesion molecules was mainly regulated by nuclear factor-${\kappa}B$ (NF-${\kappa}B$) in endothelial cells. Furthermore, P. gingivalis LPS significantly increased leukocyte adhesiveness to microvascular endothelial cells and to aortic endothelium. Taken together, our results demonstrate that P. gingivalis LPS activates microvascular endothelial cells through NF-${\kappa}B$-dependent expression of cell adhesion molecules.

DnaJC18, a Novel Type III DnaJ Family Protein, is Expressed Specifically in Rat Male Germ Cells

  • Gomes, Cynthia;Soh, Jaemog
    • Development and Reproduction
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    • v.21 no.3
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    • pp.237-247
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    • 2017
  • Mammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The full-length DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachytene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.

Cloning and characterization of the multiprotein bridging factor 1 (YIMBFI) gene from the dimorphi yeast Yarrowia lipolytica

  • Kim, Jeong-Yoon;Kim, Jang-Hwan;Cheon, Seong-Ah;Yunkyoung Song
    • Journal of Microbiology
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    • v.40 no.2
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    • pp.173-177
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    • 2002
  • In order to identify Yawowia lipolytica genes induced by serum, cDNA representational difference analysis was performed using a PCR-select CDNA subtraction method. One of the genes cloned from the subtraction was a gene (YIMBFl) homologous to Saccharomyces cerevisiae MBF1 encoding the coactivator multiprotein bridging factor 1. Disruption of YIMBFl revealed that the gene was net essential for viability, and the Ylmbf△ strain did not show any distinct phenotypic change on solid serum medium. In liquid medium, however. a difference was found in the ability to maintain hyphae induced by serum. This result suggests that the YIMbf1 protein may mediate transcriptional activation of certain genes involved in the hypha fonmation of Y. lipolytica.

Isolation of a Hypoxia/Reoxygenation Regulatory Factor in Rat Astrocytes (흰쥐 성상세포에서 산소농도의존성 유전자의 분리)

  • Park Jeong-Ae;Song Hyun-Seok;Lee Hye-Shin;Kim Kyu-Won
    • YAKHAK HOEJI
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    • v.50 no.2
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    • pp.124-128
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    • 2006
  • Astrocyte has emerged as an active regulator of brain function, which connects between blood vessels and neurons as well as is a structural component of the blood-brain barrier, From its structural characteristics, astrocyte seems to sensitively respond to oxygen tension, and, in turn, generate diverse cellular cascades. Therefore, to reveal astrocytlc events by oxygen change, we screened genes whose expressions are upregulated under reoxygenation after hypoxic stress using cDNA representational difference analysis (RDA) technique. Meteorin that regulates glial differentiation was isolated from primary cultured rat astrocytes as a hypoxia/reoxygenation regulatory factor. We cloned rat version of Meteorin (rMe-teorin) and determined full-size sequences of rMeteorin. In addition, RT-PCR analysis revealed that Meteorin was increased under reoxygenation in astrocytes and highly expressed in the developing brain. Collectively, these results suggest that Meteorin may regulate astrocyte-mediated effects in response to the change of oxygen tension in the pathophysiological states.

Genes Associated with Radiation Adaptive Response Induced by Low Level Radiation from $^{99m}Tc$ in Human Cell Lines (인체세포주에서 저선량 $^{99m}Tc$에 의해 발현되는 방사선 적응반응에 관련된 유전자에 관한 연구)

  • Kwon, An-Sung;Bom, Hee-Seung;Choi, Chan;Kim, Ji-Yeul;Lim, Wook-Bin
    • The Korean Journal of Nuclear Medicine
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    • v.35 no.5
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    • pp.313-323
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    • 2001
  • Purpose: The purpose of this study was to search activated genes that could be related to radiation adaptive response (RAR) induced by low-level radiation from $^{99m}Tc$ in human cell lines. Methods: We used gene discovery array (GDA) and representational difference analysis (RDA) methods. $^{99m}Tc$-pertechnetate was added to $2{\times}106/mL$ NC-37 cells (human lymphoblastic cells) to make concentrations ranging from 148 MBq/mL to 148 Bq/mL by serial 10 fold dilutions. After 44 hours, 2 Gy gamma irradiation was given to them using a Cs-137 cell irradiator. Results: As compared to the control (Con) group to which no $^{99m}Tc$ was added, those cells to which 148 and 14.8 KBq of $^{99m}Tc$ were added showed significantly lower damage to chromosomes, which was evaluated by metaphase analysis. Cells with 148 KBq $^{99m}Tc$ (T148 group) showed most significant protection. Activated genes in the T148 group as compared to Con group were evaluated by GDA and GDA methods. GDA revealed genes of casein kinase 2 (CK2) beta chain, immunoglobulins (lg), human leukocyte antigen (HLA)-B, and two novel genes. Twenty RAR related clones were selected by RDA method. The size of those genes was from 234 to 603 base pairs. Conclusions: RAR was induced by low dose irradiation from $^{99m}Tc$ in NC-37 cell lines. Genes related to the response included CK2, lg, HLA-B in human lymphoblastic cell lines.

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