PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.

Measurement of Apoptosis by Staining with 7-Amino-Actinomycin D with Concurrent Dual Color Immunofluorescence by Single Laser Flow Cytometry

Apoptosis is a crucial mechanism for the selective elimination of mammalian cells and is involved in many physiological and pathological processes. The heightened awareness of the importance of apoptosis has increased the need for rapid and quantitative methods for measurement of apoptotic changes. Recently, 7-amino-actinomycin D (7-AAD) has been introduced as a valuable fluorescent dye for assessing apoptosis by flow cytometry. When the cells are stained with 7-AAD in the concentration of 10 - 20 ug/ml, live cells are not stained (7-AAD ) and early apoptotic cells are weakly stained (7-AAD ) while late apoptotic or dead cells are stained brightly (7-AAD). On scattergram of forward angle light scatter vs. 7-AAD fluorescence, the three populations can be discriminated not only between each other but also from cell debris or clumps. The apoptotic cells, defined as 7- AAD cells, were demonstrated as apoptotic by morphological observation of the sorted cells. The 7-AAD cell fraction was also demonstrated to be parallel with subdiploid fraction of cells stained with PL However, 7-AAD cells, whose definition is based on the alteration of membrane integrity, have never been demonstrated to be subdiploid fraction by simultaneous DNA staining. Here we directly demonstrate that 7-AAD cells, defined on the scattergram of forward angle light scatter vs. 7-AAD fluorescence, are subdiploid fraction by staining with DNA dye whose fluorescence is collected after 530/30 band pass filter (FL-1). We also demonstrate the effects of 7-AAD concentration, fixation of cells, and proliferation of cells, on the fluorescence pattern, for reference during assessment of apoptosis by simple and rapid method for flow cytometric analysis of cells stained with 7- AAD. We also present a flow cytometric analysis of cells stained with 7-AAD Eor sequential change in apoptotic fraction, with concurrent dual-color immunophenotyping.

Classification of Borrelia burgdorferi Sensu Lato Isolated in Korea by Restriction Fragment Length Polymorphism

The organization of the ribosomal genes is unique in Borrelia burgdorferi in that the rrl (23S) and rrf (SS) genes are duplicated in tandem. Twelve Korean Borrelia isolates were classified by the method of rRNA gene restriction fragment length polymorphism (RFLP) and Southern hybridization. One of the isolates, KW-3, as well as the representative of ribotype group IV Ip89 appeared to have evolved within B. garinii. The size of the restriction bands of the 11 isolates showed different patterns from the USA and European isolates. The result by RFLP of rrf-rrl intergenic spacer region amplified by PCR showed a good correlation with that of Southern hybridization of rRNA gene. Therefore, the eleven Korean isolates could be classified as members of a new Borrelia group.

Cloning and Expression of the Gene Encoding the 32-kDa Protein of Rickettsia typhi

The crystalline surface layer protein (SLP) and a 28-32 kDa antigen of Rickettsia typhi were known as strong immunogens. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. Our study also showed that a 48-kDa protein reacted strongly with polyclonal antiserum of a patient with murine typhus. In this study, we produced three recombinant proteins (SLP, 32-kDa, and 48-kDa protein) in E. coli as fusion proteins with maltose binding proteins. The reactivity of these proteins with patients' sera was investigated.

Monoclonal antibodies specific to rickettsia typhi

No abstract available.

Monoclonal antibodies specific to rickettsia typhi

No abstract available.