Objective To observe the effect of focal cerebral ischemic preconditioning on the expression of glial fibrillary acidic protein (GFAP) and to investigate the significance of astrocyte activation in cerebral ischemic tolerance.Methods Thirty-six healthy male SpragueDawley rats were randomly divided into reischenmic,ischemic and control groups (n = 12 in each group) after ischemic preconditioning.The former two groups received 10 minutes middle cerebral artery occlusion (MCAO) preconditioning or sham operation 3 days before the 2-hour MCAO.The rats were killed 24 hours after the second MCAO.The control group only receivedthe two sham operations with an interval of three days.The infarct volume,histopathological changes,and GFAP expression in each group were compared.Results The infarct volume after ischemic preconditioning in the reischenmic and ischemic groups was 136.85 ± 14.51 mm3and 281.37 ± 29.93 mm3 respectively.The former was significantly reduced 53.15%compared to the latter (P =0.007).At the same time,neuronal degeneration and necrosis was reduced significantly,and GFAP expression was upregulated significantly (the mean absorbance for immunohistochemical staining in both groups was 102.66 ± 8.39 and 86.28 ± 6.19respectively,P = 0.009) after ischemic preconditioning in the reischemic group.Conclusions Focal ischemic preconditioning may induce brain ischemic tolerance and promote GFAP expression.The activation of astrocytes may be one of the mechanisms of cerebral ischemic tolerance.

Objective To investigate the effects of different CO2 pneumoperitoneum on cell cycle and cell cycle protein of a gastric cancer cell line. Methods Human gastric cancer cell line MNK-45 were exposed to 0,10,12 and 15 mm Hg CO2 pneumoperitoneum in vitro for 4 hrs. Cell cycle was measured by flowcytometry, the expression of CDK4 ,Cyclin D1、Rb and pRb was studied by Western-blot, and the binding ability of CDK4 and Cyclin D1 was evaluated by immunoprecipitation. Results The cell proliferation index in 15 mm Hg CO2 pneumoperitoneum group dropped significantly (27.4% ± 3. 7%) vs. (36. 4% ±3. 3%) ,P <0. 05, while that in other groups did not change significantly. The protein of CDK4、Cyclin D1and binding ability of Cyclin D1 and CDK4 dropped dramatically in the 15 mm Hg CO2 pneumoperitoneum group (0.71%±0.12%),(0.93% ±0.21%),(0.54%±0.11%),(0.18% ±0.02%) vs. (1.05% ±0.16%),(1.40% ±0.24%),(0.75% ±0.14%),(0.31% ±0.02%), all P<0.05. There were no changes of Rb in protein levels, while the phosphorylated Rb dropped obviously. Conclusion There was no obvious effects of clinical CO2 pneumoperitoneum on gastric cancer cells growth and proliferation.

Objective To study the effect and mechanism of human gastric cancer cell's apoptosis in simulated CO2 pneumoperitoneum environment by HIF-1α. Methods We used a closed box to simulate CO2 pneumoperitoneum environment, a standard surgical insufflator maintained pressure of the box at 0,5,10 and 15 mm Hg respectively. MKN-45 cell's HIF-1α mRNA and protein expression were observed before and after silencing cell's HIF-1α by real time RT-PCR and Western blot. Cell's bcl-2/bax was studied by immunohistochemistry before and after silencing HIF-1α. Cell's apoptosis ratio was measured using Annexin V-FITC/PI double labelled staining. Results In 15 mm Hg group, MKN-45 cell's HIF-1α mRNA and protein expression were significantly higher than control group (P<0.01 ), while the difference was not significant among 0,5,10 mm Hg group and control group (P>0.05). In 15 mm Hg CO2 pressure, cell's bcl-2/bax was obviously lower than in control group (P<0.05) and apoptosis ratio was more than control group(P<0.01). When HIF-1α was silenced, cell's bel-2/bax and apoptosis ratio weren't significantly different between 15 mm Hg group and control group (P>0.05 ). Conclusions MKN-45 cell's apoptosis did not experience any alterations under 0,5,10 mm Hg.15 mm Hg CO2 pneumoperitoneum environment enhanced cell's apoptosis probably by way of HIF-1α.

AIM: To study the characteristics of retinal detachment surgery after laser 　in situ　 keratomileusis (LASIK).METHODS: Eleven eyes of ten patients that experienced rhegmatogenous retinal detachment after LASIK procedure participated in the study. The characteristics of retinal detachment, management and complications after surgery were analyzed . RESULTS: Retinal detachment was characterized by the large percentage of multiple peripheral holes (73%) and giant tears (27%). All eyes underwent sclera buckling, and three of them combined with pars plana vitrectomy (PPV) and silicone oil tamponade. Silicone oil was removed after 1 month. Retina was reattached successfully at the first retinal detachment surgery in all eyes except one that succeeded at the fourth time. One eye of LASIK flap dehiscence and one eye of corneal subepithelial opacity occurred after surgery.CONCLUSION: Patients after LASIK should be carefully examined under pupillary dilation during follow-up. Sclera buckling is necessary to most retinal detachment after LASIK, and corneal protection is important in the treatment.

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae.The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid)at 3' end and 45 nucleotides at 5' end that annealed to sites upstream or downstream of the genomic target sequence to be deleted.After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS-1,the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS-1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.The expression of the Cre recombinase finally resulted in the removal of the marker gene,leaving behind a single loxP site at the chromosomal locus.The diploid mutant YS-1-sfa1 was generated,which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS-1.

Objective To investigate the clinicopathological features,diagnosis,treatment,prognosis and causative agent of a case of eumycetoma on the submaxilla.Methods A case of eumycetoma diagnosed in our department was assessed for its clinical and pathological features as well as mycologic and molecular identification.Related literature was reviewed.Results The patient was primarily characterized by swelling of the submaxilla,with multiple sinuses draining many black granules.Pathologic examination revealed a pyogenic granulomatous inflammation,and a number of lotus rhizome node-like hypha were observed in tissue samples through PAS staining.Sequence analysis of multiple loci of the isolates,including ITS 1,ITS2 and D1/D2,showed that it was mostly similar to Madurella mycetomatis with a homology of 97%.Conclusion This is a case ofeumycetoma on the submaxilla induced by a novel species of Madurella.

Objective To investigate Th1/Th2 pattern of response in psoriasis and to study the effects of CTLA4Ig on Th1/Th2 shift in psoriasis. Methods The levels of IL-2, IFN-?and IL-4 were assessed by ELISA in the supernatant of cultured peripheral blood mononuclear cells (PBMCs) from 33 patients with psoriasis vulgaris and 20 healthy blood donors. Cytokine synthesis was induced by activation with phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) with or without CTLA4Ig. Results Levels of IL-2, IFN-?and IL-4 were markedly increased by PBMCs from psoriatic patients incubated with SEB (P

Objective To investigate the status of expression and gene polymorphism of cytotoxic T lymphocyte-associated molecule4(CTLA4)on peripheral blood mononuclear cells(PBMCs)from patients with psoriasis.Methods Expression of CTLA4mRNA/antigen and polymorphism of CTLA4gene were analyzed in33and133patients with psoriasis confirmed clinically and/or pathologically,respectively.Expression of CT-LA4mRNA and antigen was detected by in situ hybridization and immmunohistochemistry.CTLA4exon1and3'untranslated region were amplified by polymerase chain reaction(PCR)and the amplified products were identified by single-stand conformation polymorphism(SSCP),restriction fragment length polymorphism(RFLP)or sequencing.Results Expression of both mRNA and antigen of CTLA4was significantly weaker on PBMCs induced by staphytococcal enterotoxin B(SEB)in patients with psoriasis than that in normal con-trols,without a regular pattern.The guanine49on exon1in association with fragment106bp of3'untranslat-ed region was shown to be linked to the susceptibility of psoriasis.Conclusion Defective translation and ex-pression of CTLA4take place in patients with psoriasis,which is possibly related to the polymorphism of CT-LA4.Our results suggest that CTLA4may be one of the candidate genes which cause autoimmunity in psoria-sis.

BACKGROUND:Corneal lymphangiogenesis is beneficial to the transport of corneal antigenic materials, and accelerates the process of antigen presentation, thereby playing an important role in corneal immunity. However, due to the paral el outgrowth of corneal blood and lymphatic vessels in transplanted corneas, it is often difficult to accurately evaluate the role of corneal lymphatic vessels in allograft rejection. OBJECTIVE:To explore the development of corneal lymphangiogenesis and angiogenesis in transplanted rat corneas after the blockade of vascular endothelial growth factor C (VEGF-C). METHODS:130 rats used to establish corneal al ogenic transplantation models were equally randomized into two groups:the anti-VEGF-C group and the control group. VEGF-C was blocked in the anti-VEGF-C group by intraperitoneal injection of neutralizing monoclonal anti-VEGF-C antibody every other day for 2 consecutive weeks. Meanwhile, rats in control groups received intraperitoneal injections of saline. Corneal angiogenesis and lymphangiogenesis were characterized using whole mount immunofluorescence, and the immune rejection of the grafts was evaluated by scoring the rejection index (RI). In addition, the expression of VEGF-C was examined by real-time PCR. The relationship of corneal lymphangiogenesis and angiogenesis to RI in transplanted corneas was also characterized. RESULTS AND CONCLUSION:VEGF-C expression was markedly downregulated after VEGF-C blockade. Corneal lymphangiogenesis developed in parallel with corneal angiogenesis in the control group. While there was a mild reduction in blood vessel area (BVA) and a significant decrease in lymphatic vessel area (LVA) in the anti-VEGF-C group (P<0.05). In addition, RI was positively correlated with BVA (P<0.05) and LVA (P<0.05) in the control group. However, although RI was significantly correlated with BVA (P<0.05) in the anti-VEGF-C group, the correlation between RI and LVA was not statistically significant (P>0.05). the graft survival time in the anti-VEGF-C group was significantly increased compared with that in the control group (P<0.05). Our results show that the outgrowth of lymphatic vessels is separated from that of blood vessels in transplanted corneas by blocking VEGF-C. The blockade of VEGF-C has a significant role in preventing corneal lymphangiogenesis in corneal beds, which results in higher al ograft survival rates.