Background Previous studies showed that after herpes simplex virus-1 (HSV-1) infection of the cornea,matrix metallo proteinase-2 (MMP-2) (produced by corneal cells and corneal epithelial cells) plays an important role in the development of HSK.Focal adhesion kinase (FAK)plays an important role on the expression,release and activation of MMPs.This study explored the expressions of MMP-2 and FAK,which induced by HSV-1 infected human corneal epithelial cells.Objective To investigate MMP-2 and FAK expression in HSV-1 infected human corneal epithelial cells.Methods Human corneal epithelial cells were infected with HSV-1 in vitro to establish cell model of viral infection.The expression of MMP-2 and FAK were detected by reverse transcription PCR (RT-PCR),Western blot,immunohistochemical method and immunofluorescence method at 2 hours,20 hours and 40 hours after infection.Results At 2 hours,20 hours and 40 hours after infection,the expressionis of MMP-2 mRNA and FAK mRNA were significantly increased in comparison with uninfected cells (MMP-2 mRNA:Ftime =0.968,P=0.436 ; Fgroup =47.649,P =0.000 ; Fi ion =0.757,P =0.536.FAK mRNA:Ftime =0.658,P =0.631 ; Fgroup =35.182,P=0.000;Finteraction =1.386,P=0.137).Western blot assays showed that there were no significant differences in p-FAK,FAK or MMP-2 expressions between infected cells and control cells after 2 hours (P＞0.05),but the expression levels of infected cells were significantly increased at 20 hours and 40 hours (both at P ＜ 0.05).Immunohistochemistry results showed that longer infection time was associated with an increased number of cells staining for MMP-2,FAK and p-FAK.Conclusions At the initial stage of HSV-1 infected,p-FAK plays an important role in the process of virus invading and MMP-2 activation.

The gene (eg1) encoding for novel endoglucanase 1 was cloned previously from Chinese straw mushroom Volvariella volvacea. EG1 has high thermal stability and optimal pH at neutral and shows great potential in textile and paper industry applications. To improve the expression level of EG1 in Pichia pastoris, the increasing copy number of clone, and its high cell density fermentation in 3.2L fermenter for its high-level expression were investigated in this work. By electro-transformation of pPICZalphaB-egl into GS115EG11 integrated with single copy of eg1 gene, A resistant transformant with 3.8 times higher level expression than GS115EG11 was screened from YPDSZ plate containing 2000 microg/mL of Zeocin. The effect of initial cell density, pH and methanol on its expression and biomass accumulation was evaluated in shaking culture. Optimal EG1 production was observed when initial cell density OD600 was 5.0. EG1 production and biomass accumulation did not seem to vary when cells were induced at different pH values. Both of EG1 and cell density were found to increase with higher methanol concentrations, reaching 62.48 IU/mL and 31.7 (OD600) respectively after 120 h induction with 2.0% (V/V) methanol compared to 30.24 IU/mL and 17.79 (OD60) with 0.25% methanol induction. EG1 expression was further increased by 6.4 times higher than shaking culture after 95.5 hours induction with methanol in fed-batch fermentation, so totally 34 times higher than that for GS115EG11 was achieved by screening of high Zeocin resistant clone and high cell density fermentation. The production of EG1 with 543.36IU/mL CMC activity and 8.80mg/mL protein expression was obtained in Pichia pastoris.
Cellulase ; biosynthesis ; genetics ; Cloning, Molecular ; Culture Media ; Electroporation ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Fungal Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Volvariella ; enzymology ; genetics

Baculoviridae ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; chemistry ; classification ; genetics ; metabolism

This study was aimed to investigate the expression level of CD4(+) CD25(high) T cells in peripheral blood of patients with idiopathic thrombocytopenic purpura (ITP) before and after treatment and to explore its significance in pathogenesis of ITP. The expressions of CD4(+) CD25(high) T cells in peripheral blood of 20 ITP patients before treatment, 20 ITP patients after treatment and 14 normal individuals (control) were detected by immunofluorescence and flow cytometry. The result showed that the expression of CD4(+) CD25(high) in ITP group before treatment was evidently lower than that in treated ITP group and control (p < 0.01), but there was no remarkable difference between the treated ITP group and the control group p > 0.05). In conclusion, the expression of CD4(+) CD25(high) T cells in the ITP group before treatment was evidently lower than that in the control group, which becomes higher than before when the patients showed effective response to the treatment. Correlation of CD4(+) CD25(high) T cell expression rate in peripheral blood with platelet level was positive. Therefore the expression level of CD4(+) CD25(high) T cells can be used as an effective index to judge the ITP prognosis. Further study of CD4(+) CD25(high) T cells regarded as an immune target is needed.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD4 Antigens ; immunology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Interleukin-2 Receptor alpha Subunit ; immunology ; Male ; Purpura, Thrombocytopenic, Idiopathic ; etiology ; immunology ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Young Adult

To investigate the anti-tumor and side effect of CpG ODN 1826 and CpG ODN2006 as an adjuvants on leukemic tumor in mouse-models, an acute lymphocytic leukemic tumor in mouse model was established, then inoculated inactivated L1210 cells alone or with different vaccine adjuvants were injected subcutaneously into each DBA/2 model mouse at different times. The activities of mice, the tumor formation rate and the growth status of leukemic tumor were observed. The tumor was examined by pathologic section. The results showed that the vaccine of inactivated L1210 cells and CpG ODN 1826 could decrease the leukemic tumor formation rate, slow down the growth of leukemic tumor mass in mice and obviously cause necrosis of tumor cells, but it could not prolong the life spans of the tumor-burden mice; while CpG ODN2006 could not only decrease the tumor formation rate, slow down the growth of tumor mass in mice and result in obvious necrosis of tumor cells, but also could eliminate the existing tumor mass in mice, and prolong the life spans of the tumor-burden mice. It is concluded that using CpG ODN2006 as an adjuvant enhances the anti-tumor effect against the leukemic tumor, prolong the life span of tumor-burden mice without obvious side effect.
Adjuvants, Immunologic ; therapeutic use ; Animals ; Cancer Vaccines ; therapeutic use ; Cytotoxicity, Immunologic ; immunology ; Female ; Leukemia L1210 ; immunology ; therapy ; Male ; Mice ; Mice, Inbred DBA ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; therapeutic use ; Random Allocation

This study was aimed to detect the expression of co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood of patients with idiopathic thrombocytopenic purpura (ITP), the content of platelet antibodies in serum (PAIgG), and to analyze the relationship between them and their correlation with the number of platelet in peripheral blood, so as to clarify the roles of co-stimulatory molecules in pathogenesis of idiopathic thrombocytopenic purpura and evaluation of disease status. The co-stimulatory molecules CD80, CD86 and CD137 in peripheral blood mononuclear cells (PBMNCs) of 48 ITP patients and 40 normal persons were detected by immunofluorescence and flow cytometry (FACS), PAIgG content in serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that CD80, CD86 and CD137 expression levels in ITP patients were (4.92 +/- 2.02)%, (8.68 +/- 4.25)%, (5.32 +/- 2.67)% respectively, PAIgG content was 210 +/- 3.02 ng/10(7) PA, all these of which were significantly higher than these in normal control group (2.01 +/- 0.75)%, (4.56 +/- 2.06)%, (1.37 +/- 1.25)%, PAIgG 20 +/- 1.13 ng/10(7) PA (p < 0.01). Correlation of co-stimulatory molecule expression with PAIgG content were positive (r = 0.302, p < 0.05), but correlation of co-stimulatory molecule expression with platelet number was negative (r = -0.369, p < 0.05). It is concluded that the co-stimulatory molecules CD80, CD86 and CD137 are involved in immune response and the incidence of ITP. Their over-expression closely associates with the pathogenesis of ITP and clinical status, so that correcting the abnormal expression and regulating the immune status may be one therapeutic strategy and have important clinical significance.
4-1BB Ligand ; blood ; Adult ; B7-1 Antigen ; blood ; B7-2 Antigen ; blood ; Blood Platelets ; immunology ; Case-Control Studies ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; Young Adult

Amino Acid Sequence ; Base Sequence ; Genes, Bacterial ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Staphylococcus epidermidis ; enzymology ; genetics ; beta-Lactamases ; chemistry ; genetics

OBJECTIVETo identify the parental origin of methyl-CpG-binding protein 2 (MECP2) gene mutations in Chinese patients with Rett syndrome.

METHODSSingle nucleotide polymorphisms (SNPs) in intron 3 of the MECP2 gene were analyzed by PCR and sequencing in 115 patients with Rett syndrome. Then sequencing of the SNP region was performed for the fathers of the patients who had at least one SNP, to determine which allele was from the father. Then allele-specific PCR was performed and the products were sequenced to see whether the allele from father or mother harbored the mutation.

RESULTSSeventy-six of the 115 patients had at least one SNP. Three hot SNPs were found in these patients. They were: IVS3+22C >G, IVS3+266C >T and IVS3+683C>T. Among the 76 cases, 73 had a paternal origin of MECP2 mutations, and the other 3 had a maternal origin. There were multiple types of MECP2 mutation of the paternal origin, including 4 frame shift, 2 deletion and 67 point (56C >T, 6C >G, 2A >G, 2G >T and 1A >T) mutations. The mutation types of the 3 patients with maternal origin included 2 frame shift and 1 point (C >T) mutation.

CONCLUSIONIn Chinese RTT patients, the MECP2 mutations are mostly of paternal origin.

Base Sequence ; Child, Preschool ; DNA Mutational Analysis ; Fathers ; Female ; Humans ; Male ; Methyl-CpG-Binding Protein 2 ; genetics ; Mothers ; Mutation ; genetics ; Parents ; Polymorphism, Single Nucleotide ; Rett Syndrome ; genetics

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.
Gene Library ; Humans ; RNA, Messenger ; analysis ; U937 Cells ; metabolism

OBJECTIVERett syndrome (RTT) is a neurodevelopmental disorder that represents one of the most common genetic causes of mental retardation in girls. The aim of this study was to investigate the correlation between MECP2 genotype and phenotype and thereby not only to provide assistance for clinical care, but also facilitate clinical genetic counseling.

METHODIndividual phenotype characteristic and clinical severity of 126 children with RTT diagnosed by molecular genetic methods were evaluated by using scales of Kerr et al and Scala et al. Statistical package SPSS 12.0 was used for analyses of data. Since the majority of the data were not normally distributed, non-parametric tests were used. The Kruskal-Wallis test/Wilcoxon Mann-Whitney test was employed to compare total severity phenotype scores. The Fisher exact test was used for comparing rates. Statistical significance was set at P < 0.05.

RESULTThere were no significant differences in the average overall scores for RTT patients with mutations in the region of methyl-CpG-binding domain (MBD) compared with those mutations in the transcription repression domain (TRD) and C terminal segment (CTS), also patients with nonsense mutations compared with missense mutations, frameshift mutations and large deletions (P > 0.05). The RTT patients with nonsense mutations located in the region of MBD have more severe phenotype than those with missense mutations in the same region (P = 0.016). Among p.T158M, p.R168X, c.806delG and p.R255X, there were no significant differences in the average overall scores (P > 0.05), but there were significant differences in language skill (P = 0.028) and in language impairment rate at different level (P = 0.019).

CONCLUSIONThere are relationships between MECP2 genotype and phenotype:the RTT patients with nonsense mutations located in MBD tend to develop more severe phenotype;there are significant differences in language skill and language impairment rate in the groups with p.T158M, p.R168X, c.806del and p.R255X, which had higher frequency in children below five-years of age and the p.R168X present with most severe impairment.

Adolescent ; Child ; Child, Preschool ; Female ; Genotype ; Humans ; Infant ; Methyl-CpG-Binding Protein 2 ; genetics ; Phenotype ; Rett Syndrome ; genetics