Comparison of lens oxidative damage induced by vitrectomy and/or hyperoxia in rabbits.

AIM: To compare of lens oxidative damage induced by vitrectomy and/or hyperoxia in rabbit. METHODS: Sixteen New Zealand rabbits (2.4-2.5 kg) were randomly divided into two groups (Group A, n=12; Group B, n=4). In Group A, the right eyes were treated with vitrectomy and systemic hyperoxia (oxygen concentration: 80%-85%, 1 ATA, 4h/d) (Group A-right), and the left eyes were treated with hyperoxia without vitrectomy surgery (Group A-left). Four rabbits in group B (eight eyes) were untreated as the controls. Lens transparency was monitored with a slit lamp and recorded before and after vitrectomy. After hyperoxic treatment for 6mo, the eyeballs were removed and the lens cortices (containing the capsules) and nuclei were separated for further morphological and biochemical evaluation. RESULTS: Six months after treatments, there were no significant morphological changes in the lenses in any experimental group when observed with a slit lamp. However, the levels of water-soluble proteins and ascorbate, and the activities of catalase and Na+-K+-ATPase were significantly reduced, whereas the levels of malondialdehyde and transforming growth factor ß2 (TGF-ß2) were significantly elevated, in both the cortices and nuclei of eyes treated with vitrectomy and hyperoxia. The increase in protein-glutathione mixed disulfides and the reduction in water-soluble proteins were more obvious in the lens nuclei. The levels of ascorbate in the vitreous fluid were also reduced after vitrectomy, whereas TGF-ß2 increased after vitrectomy and hyperoxia. Systemic hyperoxia exposure increased these effects. CONCLUSION: Removal of the intact vitreous gel with vitrectomy and exposing the lens to increased oxygen from the retina induce lens oxidation and aggregation. Thus, an intact vitreous gel structure may protect the lens from oxidative insult and maintain lens transparency.

Hydrogen saline prevents selenite-induced cataract in rats.

PURPOSE: The aim of this study was to investigate the potential antioxidative effect and mechanism for the protective effects of hydrogen saline on selenite-induced cataract in rats. METHODS: Sprague-Dawley rat pups were divided into the following groups: control (Group A), selenite induced (Group B), and selenite plus hydrogen saline treated (Group C). Rat pups in Groups B and C received a single subcutaneous injection of sodium selenite (25 µmol/kg bodyweight) on postnatal day 12. Group C also received an intraperitoneal injection of H2 saline (5 ml/kg bodyweight) daily from postnatal day 8 to postnatal day 17. The development of cataract was assessed weekly by slit-lamp examination for 2 weeks. After sacrifice, extricated lenses were analyzed for activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of malondialdehyde, reduced glutathione (GSH), and total sulfhydryl contents. RESULTS: The magnitude of lens opacification in Group B was significantly higher than in Group A (p<0.05), while Group C had less opacification than Group B (p<0.05). Compared with Group B, the mean activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of GSH, and total sulfhydryl contents were higher, whereas the level of malondialdehyde was lower following treatment with hydrogen saline(p<0.05). CONCLUSIONS: This is an initial report showing that hydrogen saline can prevent selenite-induced cataract in rats. It acts via maintaining antioxidant enzymes and GSH, protecting the sulfhydryl group, and inhibiting lipid peroxidation.

Oxidative responses induced by pharmacologic vitreolysis and/or long-term hyperoxia treatment in rat lenses.

PURPOSE: The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo. METHODS: Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors. RESULTS: No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis. CONCLUSIONS: Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

[Screen of a membrane protein molecule from Vero cells capable of binding Japanese encephalitis virus(JEV)].

AIM: Identification of membrane protein molecules Vero cells binding JEV. METHODS: Membrane protein extract was subject to co-immunoprecipitation (Co-IP) with JEV, and identified by mass (MS) spectrometry. The binding between specific extract protein and JEV was measured by MS, flow cytometry (FCM) and immunofluorescence assay (IFA). RESULTS: Heat shock protein 90 beta (HSP90 beta) was identified by Co-IP and MS, and its binding JEV activity was confirmed by FCM and IFA. CONCLUSION: HSP 90 beta from Vero cells membrane could bind JEV.

[Preparation, characterization and preliminary application of monoclonal antibody against human mu chain].

AIM: To prepare monoclonal antibody (mAb) against human mu chain with high titer and establish a capture ELISA for early serological diagnosis of infectious diseases. METHODS: BALB/c mice were immunized with human IgM. Hybridoma cell line which could stably secret the mAb to human IgM was established by routine cell fusion technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by indirect ELISA and Western blot, respectively. A capture ELISA was established by using purified mAb to capture specific IgM for early diagnosis of Japanese encephalitis. RESULTS: One hybridoma cell line 2E5 stably secreting mAb against human IgMmu chain was obtained. The titer of ascites of the mAb was 1 x 10(-6) and the Ig subclass was IgG1(kappa). Relative affinity of 2E5 was 1 x 10 (-5). Western blot analysis showed that mAb 2E5 reacted specifically to mu chain. Both sensitivity and specificity of the capture ELISA in detecting specific IgM in Japanese encephalitis patients sera were high. CONCLUSION: mAb 2E5 against human mu chain was prepared successfully, and a capture ELISA for early serological diagnosis of Japanese encephalitis was set up.

[Preparation of specific monoclonal antibody against nucleocapsid protein of SARS coronavirus].

AIM: To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein. METHODS: N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. RESULTS: DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the M(r) of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as primary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein. CONCLUSION: The prepared recombinant N protein and mAbs against N protein lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.

[Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris].

AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.