Application of truncated immunodominant polypeptide from hepatitis E virus (HEV) ORF2 in an assay to exclude nonspecific binding in detecting anti-HEV immunoglobulin M.

The diagnosis of recent hepatitis E virus (HEV) infection depends on serologic testing for anti-HEV IgM; however, false-positive results may occur. In the present study, we cloned the ORF2 fragment of genotype 4 HEV and demonstrated that a subregion covering amino acids 459 to 607 in ORF2 forms the immunodominant B-cell epitopes, as it does in genotype 1 viruses. Truncation of several residues from either the N or C terminus of the polypeptide abolished the reactivity of anti-HEV from naturally infected persons. By the combination of high reactivity of the immunodominant polypeptide and poor reactivity of the truncated polypeptide, we established an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-HEV IgM. In this assay, all 37 sera that were HEV RNA positive reacted with the immunodominant polypeptide but not with the truncated one, and none of 159 sera from healthy persons reacted with either of the polypeptides. In retesting of 117 sera that originally tested positive for anti-HEV IgM, using a Genelabs kit, only 34 were positive and 83 were negative. Western blot analyses and other experiments strongly indicated that these 83 discordant sera were negative for anti-HEV IgM. Furthermore, among the 117 sera, 5 reacted with both the immunodominant and truncated polypeptides, with comparable optical densities at 450 nm. However, their reactivity was demonstrated to result from nonspecific binding. Together, the data indicate that the poor reactivity of a truncated ORF2 polypeptide can be used to exclude nonspecific binding in the detection of anti-HEV IgM.

[Effects of inhibition of ANG-2 expression in Ishikawa cell line].

OBJECTIVE: To investigate the effect of RNA interference mediated angiopoietin-2 (ANG-2) gene silencing on human endometrial carcinoma cell line Ishikawa. METHODS: Short hairpin RNA (shRNA) targeting ANG-2 gene was designed and transfected into Ishikawa cells with Lipofectamine 2000. The mRNA and protein expression level of ANG-2, proliferation, morphological changes, apoptosis, cell cycle and invasive ability of the cells after transfection were analyzed. RESULTS: The shRNA targeting the human ANG-2 gene effectively decreased the expression of ANG-2 on both mRNA and protein level, the proliferation inhibition rate of the Ishikawa cells was 63.11%, cell apoptosis was induced, and the cell cycle was arrested in G1 phase. The apoptotic rate of the Ishikawa cells in the transfected group was significantly higher, and the invasive ability was decreased markedly, than that of control groups. CONCLUSION: The shRNA targeting human ANG-2 gene could reduce ANG-2 expression, inhibit cellular growth and invasion in Ishikawa cells in vitro.

Combined treatment of ionizing radiation with genistein on cervical cancer HeLa cells.

The anticancer agent genistein inhibits cell growth of tumor cell lines from various malignancies. In our study, we investigated the effectiveness of combined treatment of ionizing radiation (IR) with genistein on cervical HeLa cells and its possible mechanism. It was found that the inhibitory rate in cells with combined treatment was significantly higher than that of the cells treated with IR or genistein alone. After treatments of IR (4 Gy) combined with genistein (40 micromol/L), the apoptotic index of the cells was significantly increased and the cells were arrested in the G2/M phase. Survivin mRNA expression increased after IR (4 Gy), while it significantly decreased after combined treatment. These findings indicated that genistein enhanced the radiosensitivity of cervical cancer HeLa cells, and the mechanisms for this action might include increase of apoptosis, decrease of survivin expression, and prolongation of cell cycle arrest.

Inhibitive effect of genistein on hypoxia-induced basic fibroblast growth factor expression in human retinal pigment epithelium cells.

AIM: The time course changes of basic fibroblast growth factor (bFGF) expression induced by hypoxia and the effects of genistein on hypoxia-induced bFGF expression in the human retinal pigment epithelium (RPE) cells were studied. METHODS: The bFGF mRNA expression was examined by reverse transcription polymerase chain reaction. The bFGF protein expression was detected by Western blot. RESULTS: Hypoxia significantly increased bFGF mRNA expression. The maximal level detected at 24 h was approximately two times that at the start of treatment. With pretreatment of genistein (10, 20, 50, 100, and 200 microM) for 30 min, the elevated expression of bFGF mRNA was suppressed in a concentration-dependent manner. bFGF mRNA expression was reduced to 30.4% by 200 microM of genistein when compared with that untreated with genistein. Hypoxia treatment also remarkably increased the expression of bFGF protein. At 24 h after hypoxia, when the highest expression of bFGF protein was observed, it was approximately two times as much as that at the start of treatment. Genistein (10, 20, 50, 100, and 200 microM) could also suppress bFGF protein expression in a concentration-dependent manner. The highest suppression was observed when exposed to 200 microM of genistein, which was 43% of control. CONCLUSIONS: These results suggested that suppression of bFGF expression in RPE cells might partly account for the inhibitive effect of genistein on retinal neovascularization in vivo.

Genistein inhibited retinal neovascularization and expression of vascular endothelial growth factor and hypoxia inducible factor 1alpha in a mouse model of oxygen-induced retinopathy.

The effects of genistein on neovascularization, vascular endothelial growth factor (VEGF), and hypoxia inducible factor 1alpha (HIF1alpha) protein expression in a mouse model of oxygen-induced retinopathy were studied. The model of oxygen-induced retinal neovascularization was induced in newborn C57BL/6 mice by exposing 7-day-old mice to 75% oxygen for 5 days and then housing them in room air (relative hypoxia). Retinopathy was assessed by quantitation of vascular cell nuclei anterior to inner limiting membrane. Judged by relative fluorescence using a confocal scanning laser microscope coupled to a computer, VEGF and HIF1alpha protein expression were investigated. Genistein markedly inhibited the numbers of nuclei protruding above the inner limiting membrane under relative hypoxia conditions. The levels of nuclei numbers were suppressed by 50, 100, and 200 mg/kg body weight /day genistein to 87.4%, 72.0%, and 59.4%, respectively, compared to that untreated with genistein. VEGF protein was constitutively expressed in the preretinal area under normoxia conditions. Genistein markedly inhibited relative-hypoxia-elicited VEGF expression elevation in a dose-dependent manner. HIF1alpha expression was also observed in normoxia conditions. There was a 2.4-fold induction in preretinal HIF1alpha expression in oxygen-reared animals when compared to room-air-reared animals. Genistein dose-dependently suppressed HIF1alpha protein expression. These results indicated that the inhibition of VEGF and HIF1alpha protein expression by genistein may partly account for its effect on retinal neovascularization in vivo, and genistein could be an effective agent in the prevention and treatment of ocular neovascularization.