Biomedical subjects

H Poo

Publications and source records attributed to H Poo.

5 recordsLinked to original sources

PubMed1998

Establishment and characterization of cell lines constitutively expressing hepatitis B virus X-protein.

We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.

Carcinoma, Hepatocellular↗

PubMed1995

Interaction of Fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

3T3 Cells↗

PubMed1994

Ligation of CD3 triggers transmembrane proximity between LFA-1 and cortical microfilaments in a cytotoxic T cell clone derived from tumor infiltrating lymphocytes: a quantitative resonance energy transfer microscopy study.

We have explored the transmembrane associations of leukocyte function associated antigen-1 (LFA-1) in response to T cell receptor ligation using resonance energy transfer (r.e.t.) microscopy to detect receptor to microfilament proximity. R.e.t. was detected using both imaging and photon counting techniques. T cells were labeled with fluorescein-conjugated F(ab')2 fragments of an anti-LFA-1 monoclonal antibody. Cells were incubated at 37 degrees C on unmodified glass surfaces and surfaces coated with anti-CD3 or anti-H-9 antibodies. Microfilaments of fixed cells were labeled with rhodamine-phalloidin. R.e.t. was not affected on unmodified (blank) or irrelevant antibody-treated (H-9) surfaces. However, both fluorescence images and photon count rates were significantly enhanced when cells bound to anti-CD3-coated surfaces. This enhancement was not due to a general effect of T cell activation on transmembrane cytoskeletal proximity since CD45-phalloidin r.e.t. was not affected by CD3 ligation. These experiments provide direct physical evidence that ligation of the CD3 complex specifically increases the proximity of LFA-1 and microfilaments, which may be relevant to T cell mediated adherence reactions.

Actin Cytoskeleton↗

PubMed1992

Surface-bound immune complexes trigger transmembrane proximity between complement receptor type 3 and the neutrophil's cortical microfilaments.

To characterize the transmembrane associations participating in antibody-dependent binding, we have used resonance energy transfer (r.e.t.) microscopy to assess the molecular proximity of complement receptor type 3 (CR3) and Fc gamma R type II (Fc gamma RII) and type III (Fc gamma RIII) with microfilaments during neutrophil adherence to untreated surfaces, surfaces coated with BSA, surfaces coated with BSA/anti-BSA F(ab')2 complexes, or surfaces coated with BSA/anti-BSA rabbit IgG immune complexes. Receptors were labeled with fluorescein-conjugated antireceptor Fab fragments, whereas microfilaments were labeled with rhodamine-phalloidin. CR3-to-microfilament r.e.t. was dramatically increased in neutrophils adherent to IgG immune complex-coated surfaces but not untreated or control surfaces. However, the low level of r.e.t. between donor-labeled anti-Fc gamma RII Fab fragments and rhodamine-phalloidin was not affected by any condition including surface-bound immune complexes. Significant r.e.t. levels between Fc gamma RIII and microfilaments were not found under any condition. We suggest that CR3 plays an important role in tethering surface-attached immune complexes to the neutrophil's cytoskeleton.

Actin Cytoskeleton↗

PubMed1989

Transformation of a filamentous cyanobacterium by electroporation.

The filamentous cyanobacterium Anabaena sp. strain M131 was transformed with the shuttle vector pRL6 by electroporation. Optimum conditions for electroporation required relatively high field strengths with short time constants. Restriction significantly lowered the efficiency of transformation. A plasmid containing a single unmodified AvaII restriction site transformed cells with about 100-fold-lower efficiency than did the same plasmid with a modified restriction site.

Cell Membrane Permeability↗