Bei Chang Yang

One-step transformation of yeast in stationary phase.

SummaryA fast yeast-transformation technique has been developed by adding thio compounds to alkali-ion based protocols and incubating at 45°C. This procedure is especially recommended for cells from stationary phase at a density up to 2.5×108 cells/ml. It involves only one step for the preparation and transformation of competent cells within 30 min. The yield was more than 104 transformants/μg plasmid DNA. This protocol is easy to scale up for many DNA samples and is also applicable for yeast cells from different types of storages.

Enhanced interleukin-4 expression in patients with endometriosis

OBJECTIVE To investigate the expression of cytokines by T-helper cells type 1 (interleukin [IL]-2 and interferon-gamma [IFN-gamma]) and type 2 (IL-4 and IL-10) in peripheral blood monocytes and peritoneal fluid (PF) cells from patients with endometriosis. DESIGN Peripheral blood (PB) monocytes and PF cells from patients with endometriosis were isolated and the expression of cytokines investigated. SETTING Institute for the treatment of endometriosis and research institute in university-based medical center. PATIENT(S) The study included 7 women with normal pelvic structure, 36 patients with endometriosis, and 7 women with pelvic adhesion but without apparent endometriotic lesion. The existence and severity of endometriosis was determined by laparoscopic examination. INTERVENTION(S) All patients received laparoscopic operation to identify the existence and stages of endometriosis. Danazol (200 mg, twice daily) was prescribed for those with endometriosis. MAIN OUTCOME MEASURE(S) Transcription of cytokines were directly analyzed using the reverse transcriptase-polymerase chain reaction method. The concentrations of cytokines in peritoneal fluids and sera were analyzed by the ELISA. RESULT(S) Levels of IL-4 messenger RNA (mRNA) and protein in the PB and peritoneal cells of patients with endometriosis were higher than those of normal patients, whereas levels of IFN-gamma mRNA and protein were suppressed. There were no significant changes in the mRNA or protein levels of IL-2 or IL-10 in both peritoneal fluids and sera. The level of IL-4 was reduced to normal after combined treatment with laparoscopic surgery and danazol. The secretion of IFN-gamma was elevated after treatment. CONCLUSION(S) Cytokine secretion by T-helper cells type 1 and type 2 is altered in women with endometriosis, suggesting that these T-helper subsets play a role in the immunologic responses to endometriosis.

The expression of soluble intercellular adhesion molecule-1 in endometriosis

OBJECTIVE To investigate the association between concentrations of soluble intercellular adhesion molecule-1 (ICAM-1) and interferon-gamma (IFN-gamma) with regard to the severity of endometriosis. DESIGN Cross-sectional study. SETTING Infertility unit in a university hospital. PATIENT(S) Seventy-one infertile patients who underwent laparoscopic evaluation were divided into two groups: 36 patients with endometriosis served as cases, and 35 patients without endometriosis served as control subjects. INTERVENTION(S) Peripheral blood and peritoneal fluid (PF) were collected from these patients during laparoscopic examination. MAIN OUTCOME MEASURE(S) Quantitative determination of levels of human ICAM-1 and IFN-gamma was performed with the use of an ELISA. The data were evaluated by Students t-test, analysis of variance, and regression analysis. RESULT(S) Significantly elevated levels of soluble ICAM-1 were found in the sera of patients with endometriosis, especially those with advanced stages of the disease. Decreased IFN-gamma levels, which were negatively correlated with serum levels of soluble ICAM-1, also were noted in the PF of patients with endometriosis. CONCLUSION(S) The increased serum levels of ICAM-1 found in patients with endometriosis may indicate the presence of an active disease process. Further, the increased levels of soluble ICAM-1 in peripheral blood were inversely correlated with the concentrations of IFN-gamma in PF. The increased levels of soluble ICAM-1 in plasma may be associated with an immunologic feedback response that blocks further infiltration of immune cells. These findings may be of value in the diagnosis and evaluation of endometriosis.

Fas Ligand on Tumor Cells Mediates Inactivation of Neutrophils

The expression of Fas ligand (FasL) on tumor cells (tumor FasL) has been implicated in their evasion of immune surveillance. In this study, we investigated the cellular mechanism for FasL-associated immune escape using melanoma B16F10-derived cells as a model. Transfectants carrying FasL-specific ribozymes expressed low levels of FasL (FasLlow tumor cells) as compared with those carrying enhanced green fluorescent protein-N1 plasmids (FasLhigh tumor cells). When injected s.c. into C57BL/6 mice, FasLlow tumor cells grew more slowly than did FasLhigh melanoma cells. FasLhigh tumor cells showed more intensive neutrophilic infiltration accompanied by multiple necrotizing areas than did FasLlow tumor cells. The average size of FasLlow tumors, but not of FasLhigh tumors, was significantly enhanced in mice depleted of neutrophils. Consistently, a local injection of LPS to recruit/activate neutrophils significantly delayed tumor formation by FasLlow tumor cells, and slightly retarded that of FasLhigh tumor cells in both C57BL/6 and nonobese diabetic/SCID mice. Neutrophils killed FasLlow melanoma cells more effectively than FasLhigh melanoma cells in vitro. The resistance of FasLhigh melanoma cells to being killed by neutrophils was correlated with impaired neutrophil activation, as demonstrated by reductions in gelatinase B secretion, reactive oxygen species production, and the surface expression of CD11b and the transcription of FasL. Local transfer of casein-enriched or PMA-treated neutrophils delayed tumor formation by melanoma cells. Taken together, inactivation of neutrophils by tumor FasL is an important mechanism by which tumor cells escape immune attack.

Functional expression of rat M5 muscarinic acetylcholine receptor in yeast.

We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast alpha-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [3H]-N-methyl scopolamine ([3H]NMS) with a kd of 22.77 nM and Bmax of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on alpha-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.

Antioxidant administration inhibits exercise-induced thymocyte apoptosis in rats.

PURPOSE The purpose of this study was to investigate the effect of antioxidant on exercise-induced apoptosis in rat thymocytes. METHODS After exercise at 13.8 m x min(-1) for 60-90 min x d(-1) on a motor-driven drum exerciser for 2 consecutive days, rat thymocyte apoptosis was monitored by the feature of DNA fragmentation. To study the effect of antioxidant, rats were administered with butylated hydroxyanisole (BHA) for 7 d before exercise. RESULTS Exercise could induce thymocyte DNA fragmentation as detected on electrophoretic gel and by cell death detection ELISA kit. Further studies indicated that pretreatment with antioxidant BHA to rats resulted in a blockage of exercise-induced DNA fragmentation. The concentrations of glutathione (GSH) were not significantly changed in rat thymocytes after exercise with or without BHA treatment. CONCLUSION These results suggest that reactive oxygen species may play a role in thymocyte apoptosis induced by exercise. However, changes in GSH levels were not observed in this exercise model.

The differential expression of intercellular adhesion molecule-1 (ICAM-1) and regulation by interferon-gamma during the pathogenesis of endometriosis

Problem:  To establish an in vitro culture model of the endometrium and endometriotic lesions, and demonstrate the different expressions of intercellular adhesion molecule‐1 (ICAM‐1) in these lesions.

Attenuation of house dust mite Dermatophagoides farinae-induced airway allergic responses in mice by dehydroepiandrosterone is correlated with down- regulation of TH2 response

Dehydroepiandrosterone (DHEA), an abundant androgen in circulation, has important immunomodulating effects on T‐cell differentiation; however, it is not known whether this hormone influences allergic responses.

Coexposure of lead- and lipopolysaccharide-induced liver injury in rats: Involvement of nitric oxide-initiated oxidative stress and TNF-α

In this study, we investigated the interaction between lipopolysaccharide (LPS) and lead (Pb) and the involvement of tumor necrosis factor-α (TNF-α) and oxidative stress in Pb-plus-LPS (Pb/LPS)-induced liver damage in rats. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), TNF-α, nitric oxide (NO), and lipid peroxidation (LPO) were determined in rats treated with Pb and/or LPS. Pb ranging from 0 to 15 mg/kg dose dependently increased AST, ALT, NO, or LPO in LPS-treated rats. Pretreatment with iNOS inhibitor 1400W reduced NO, LPO, TNF-α, AST, and ALT in Pb/LPS-treated rats. Thus, Pb increased LPS-induced liver damage, which might be associated with increased NO-initiated oxidative stress and TNF-α in rats.

EXOGENOUS DEHYDROEPIANDROSTERONE MODIFIED THE EXPRESSION OF T HELPER-RELATED CYTOKINES IN NZB/NZW F1 MICE

The onset of lupus-like disease in NZB/NZW F1 mice was correlated with the expression of IL-10 at 4 m of age, and with a sequential enhanced expression of IFN-gamma and IL-6 between 6 to 8 m of age. The expression of IFN-gamma and IL-6 was associated with exacerbation of disease symptom, production of anti-DNA antibody, and increase in total serum IgG1. Exogenous dehydroepiandrosterone (DHEA) given in animal diet significantly prolonged survival, and delayed formation of autoantibody of NZB/NZW F1 mice as compared to mice fed on control diet. The effect of DHEA paralleled a delay in the expression of IL-10 and IL-6 and an earlier detection of IL-12 transcripts. Moreover, DHEA-fed mice had higher serum IgG2a level than control diet-fed mice. Collectively, DHEA may modify the activation of distinct subset of T helper cells in NZB/NZW F1 mice at different phases of disease progression.