Biomedical subjects

M F Leung

Publications and source records attributed to M F Leung.

23 records · Page 2Linked to original sources

PubMed1990

Gene construction and mutagenesis for site specific modification of protein with carbohydrate.

We reported the construction of the structural gene for trans-activator (Tat) protein of human immunodeficiency virus. While maintaining the same amino acid sequence as the viral protein, the corresponding nucleotide sequence was modified to create additional recognition sites for restriction endonucleases and to prevent basepair mismatch during gene assembly. The oligonucleotides were synthesized chemically, purified and assembled into five gene blocks. The gene blocks were cloned into plasmid vectors and later reassembled into a complete gene. The use of gene blocks facilitated in vitro mutagenesis by the cassette mutagenesis method.

Amino Acid Sequence↗

PubMed1989

Relationship between 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione.

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthionine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 microM 1-chloro-2,4-dinitrobenzene and 250 microM buthionine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthionine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 microM 2-cyclohexene-1-one, which depletes free glutathione by conjugation, resulted in a complete depletion of total cellular glutathione content without altering the microtubule organization. These results suggest that the total glutathione content may be important for cellular recovery from 1-chloro-2,4-dinitrobenzene-mediated cytoskeletal injuries, and that microtubule disassembly observed in 1-chloro-2,4-dinitrobenzene-treated cells probably results from depletion of cellular glutathione coupled with binding to tubulin and/or other microtubule components.

Animals↗

PubMed1987

Biological properties of suppressive E-receptor factor on lymphokine function.

A potent immunosuppressive factor isolated from malignant ascites fluids showed serological cross-reactivity with the E-receptor of human peripheral blood T lymphocytes. Thus, this factor was named suppressive E-receptor (SER) factor. In this study, we examined the effect of this immunosuppressor, SER, on lymphokine functions of human mononuclear cells participating in polyclonal T cell activation. SER is active at nanomolar concentrations in vitro and the inhibitory effect of SER was most pronounced when added at the initiation of stimulation with phytohemagglutinin or anti-T3 antibody. Concomitant with the inhibition on PHA-induced DNA synthesis, lymphocytes that were treated with SER failed to progress beyond G1 phase of cell cycle. These growth-arrested cells did expire after 7 days of culture in vitro. This anti-proliferative effect of SER was more easily demonstrated with normal lymphoid cells in culture than transformed cells or fibroblast cells. SER effectively interfered with the lympho-proliferative properties of interleukin 2 (IL 2) on human peripheral blood mononuclear cells and an IL 2-dependent murine cytotoxic T cell line. However, excess quantities of exogenous IL 2, especially when added in conjunction with IL 1, were able to partially overcome the ability of SER to inhibit T cell proliferation. In contrast to the inhibition on DNA synthesis of human lymphoblasts, expression of IL 2 receptor was only minimally inhibited by SER during the first 24 h of culture (24% inhibition at 12 h and 34% inhibition at 24 h) but it was followed by full expression of IL 2 receptor by 48 h. Thus, SER merely reduced the rate of expression of IL 2 receptor and was not able to inhibit the transcription of new message from activated T lymphocytes. Taken together, these studies indicate that SER acts as a noncytolytic anti-proliferative factor on immune responses that are mediated by T cells. SER appears to act on a relatively late event during T cell activation, perhaps on some portion of the DNA replication pathway.

Cell Line↗

PubMed1979

Distinctive RNA transcriptase, polyadenylic acid polymerase, and polyuridylic acid polymerase activities associated with Pichinde virus.

Three RNA polymerase activities were found and associated with purified Pichinde virus, a member of the Arenaviridae. A heat-labile polymerase activity which required all four ribonucleoside triphosphates for optimal activity co-sedimented on sucrose gradient centrifugation with the viral ribonucleoprotein complex from detergent-disrupted virus preparations. This enzyme synthesized heteropolymers which represented about 23% of the genome RNA as determined by nucleic acid hybridization. Two relatively heat-stable polymerase activities which differed in their cation requirement and substrate specificity were recovered with the virus-associated ribosomes. These polymerase activities synthesized homopolymers of limited chain length: in the presence of 10 mM Mg2%, polyuridylic acid was made, whereas in the presence of 1 mM Mn2%, polyadenylic acid was made. The addition of complementary RNA synthesized with the viral transcriptase in vitro to the reaction mixture containing the polyadenylic acid polymerase activity resulted in the terminal addition of polyadenylic acid to the complementary RNA. The possible function of the ribosome-associated polymerase activities in the replication of the virus is discussed.

Animals↗

PubMedDate not supplied

Pathogenesis of IgA nephropathy in ethanol consumption: animal model and cell culture studies.

Using the intragastric ethanol infusion model of IgA nephropathy, we investigated the hypothesis that in this model mesangial changes commence prior to the deposition of IgA. We studied the two cellular components of the glomerular mesangium: the mononuclear phagocyte and the contractile mesangial cell. In the in vivo model, we observed a mononuclear phagocyte influx in the mesangium of alcoholic rats before the deposition of IgA. Using molecular techniques on cultured contractile mesangial cells, we demonstrated a threefold increase in interleukin-6 mRNA expression in contractile cells incubated with ethanol. These mesangial changes in the cellular composition, and in the autocrine cytokine system, suggest a direct role for ethanol in the pathogenesis of IgA nephropathy.

Alcohol Drinking↗