BACKGROUND:The linkage and synergistic effect of adaptor proteins can effectively regulate signal transduction of T cel s, which can form a limit or amplification cascade to realize the complex immune function of T cel s. C-terminal Src kinase (Csk)-binding protein (Cbp) is an adaptor protein, which mainly exert the negative feedback regulation of Src kinase activity. This negative feedback effect depends on Y317 of Cbp, which may be involved in the SH2 domain of Csk. OBJECTIVE:To explore the effects of high expression of Cbp on ultrastructure and related biological function of Jurkat cel s. METHODS:The virus particles were constructed with expressing enhanced green fluorescent protein (EGFP) only and Cbp-EGFP fusion protein to transfect Jurkat cel s. There were untransfected group (Jurkat group), negative control group (transfected with expression of EGFP virus only), and Cbp group (transfected with Cbp-EGFP virus). RESULTS AND CONCLUSION:Confocal microscope showed that cel transfection efficiency was more than 95%and Cbp was located on the cel membrane. Optical microscope showed after transfection with Cbp-EGFP virus, more Jurkat cel s shrunk, with poor size uniformity. Apoptosis detection showed that after transfection with Cbp-EGFP virus, the number of apoptotic and necrotic cel s was greatly increased. Cbp mRNA expression was increased, Csk expression was decreased obviously and lymphocyte-specific protein tyrosine kinase expression was increased. So, in Jurkat cel s, the high expression of Cbp can decrease the uniformity of cel s and increase the necrosis cel s, thus inhibiting the signal transduction.

OBJECTIVETo construct a modified and enhanced thymidine kinase (TK) vector regulated by human telomerase catalytic subunit promoter (hTERT) promoter and cytomegalovirus (CMV) enhancer and its killing effect on nasopharyngeal carcinoma in vitro and in vivo and its safety in vivo.

METHODSThe pGL3-basic, as basic vector template, was linked and constructed into TK vector regulated by hTERT promoter and CMV enhancer with mono-promoter vector as control. Enhanced TK expression was confirmed by fluorescent microscopy and real time fluorescent quantitative PCR. Telomerase activity was measured by stretch PCR. Tumour killing effects were examined by MTT and Boyden areola. The effects of enhanced TK on the invasiveness of tumor cell NPC 5-8F and the growth of xenograft implanted in nude mice were investigated.

RESULTSCompared with non-enhanced vector, TK expressed by the enhanced vector significantly increased in NPC 5-8F and MCF-7 cells, telomerase activity was positive in human in NPC 5-8F cells and breast cancer MCF-7 cells and negative in control human blood vessel endothelium ECV-304 cells. After ganciclovir(GCV) treatment, NPC 5-8F cell survival rate and invasiveness decreased and tumor progress of NPC xenograft implanted in nude mice was inhibited, without obvious toxicity effects on mouse liver and kidney.

CONCLUSIONSThe enhanced TK vector regulated by hTERT promoter and CMV enhancer can obviously and specifically inhibit and kill nasopharyngeal carcinoma cells in culture and nasopharyngeal carcinoma xenograft in nude mice in vivo, without obviously toxic side effects on nude mice. The targeted and enhanced TK gene vector with high performance may be a new tumour targeted gene therapy strategy clinically to aim directly at most malignant tumours including nasopharyngeal carcinoma, with more extensive anti-cancer spectrum.

Animals ; Cell Line, Tumor ; Cell Survival ; Cytomegalovirus ; genetics ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nasopharyngeal Neoplasms ; therapy ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Telomerase ; genetics ; Thymidine Kinase ; genetics ; Transfection

The complete nucleotide sequence of a cDNA clone encoding weever ribosomal protein L8, WeevL8, is described here. The WeevL8 cDNA has 848 nucleotides in full length and encodes a 257 amino-acid protein with a calculated molecular mass of 28.02 kDa. Two conserved domains have been identified in WEEVL8. It was concluded fromsequence alignment that WeevL8 gene was quite conservative, consistent with its role as a house-keeping gene. A phylogenetic analysis made by L8 protein showed the similar phylogenetic relationship to that with 18s rDNA. The high similarity supports the notion that ribosomal protein L8 can also be used as phylogenetic criterion.

To characterize the complete genome sequence of coxsackievirus B1 (CVB1) MSH/KM9/2009 strain isolated from Yunnan, China,2009. Eight overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were compared with other known CVB1 strains. The genome of the CVB1 MSH/KM9/2009 strain had 7384 nucleotides in length, and contained a 741nt non-translated region (NTR) at the 5' end and a 94nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion, but some changes of amino acid were unique. The complete genome sequence alignments showed that the CVB1 isolate MSH/KM9/2009 strain shared the highest nucleotide (80.9%, 81.6%, 80.5% and 80%) and amino acid (95.6%, 95.8%, 96.2% and 95.6%) identities to the CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain, respectively. Phylogenetic tree analysis showed that the MSH/KM9/2009, CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain clustered into same group. The newly isolated CVB1 strain MSH/KM9/2009 from Yunnan Province belonged to genotype CVB1.
Base Sequence ; Child, Preschool ; China ; Coxsackievirus Infections ; virology ; Enterovirus ; classification ; genetics ; isolation & purification ; Female ; Genome, Viral ; Genotype ; Humans ; Infant ; Male ; Open Reading Frames ; Phylogeny ; Viral Proteins ; genetics

The complete nucleotide sequence of two human enterovirus 71 strains (KMM09 and KM186-09) isolated in Yunnan,China, were determined by RT-PCR and sequencing. As with other human enteroviruses, the genomes were 7 409 nucleotides (nts) in length and encoded 2 193 aa. Phylogenetic analysis based on VP1 regions revealed that the two isolates belonged to subgenotype C4a. In structural genomic regions, subgenotype C4 was most homologous to other strains of C genotype when compared to other genotypes. In non-structural genomic regions, subgenotype C4 was more homologous to CA16/G10 and other strains of B genotype than to other strains of C genotype. RDP3 and Blast analysis displayed evidence of recombination in non-structural genomic regions between subgenotype B3 and C4, C4 and CA16/G10. The full-length genome of the human enterovirus 71 strains provided an overview of the diversity of genetic characteristics of a circulatinghuman enterovirus 71.
Animals ; Base Sequence ; Cercopithecus aethiops ; China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Feces ; virology ; Genome, Viral ; Genotype ; Humans ; Molecular Sequence Data ; Phylogeny ; Vero Cells

OBJECTIVETo observe the gene and protein expression of herpes simplex virus type I-thymidine kinase (HSV(1)-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV(1)-tk gene mediated by GE7 delivery system.

METHODSGE7-polylysine/pCMV-HSV(1)-tk/polylysine-HA20 complexes were constructed. Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein, respectively. The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection. RT-PCR and Western Blot were preceeded to determine the expression of HSV(1)-tk gene and protein in the tumor tissues and other organs.

RESULTSIn the group of arterial injection with 4-element complexes, the HSV(1)-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs. In the group of arterial injection with saline buffer, no HSV(1)-tk gene and protein was detected in both tumor tissues and other organs. In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys.

CONCLUSIONHigh target and gene transfer rates can be obtained when HSV(1)-tk gene is transferred via the artery route mediated by GE7 delivery system. HSV(1)-tk protein can be expressed after the gene transfer. The results may provide a new strategy for target killing of HSV(1)-tk/GCV system in ovarian tumors.

9,10-Dimethyl-1,2-benzanthracene ; Adenocarcinoma ; chemically induced ; genetics ; metabolism ; Animals ; Female ; Gene Transfer Techniques ; Herpesvirus 1, Human ; genetics ; Infusions, Intra-Arterial ; Ovarian Neoplasms ; chemically induced ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Thymidine Kinase ; biosynthesis ; genetics

OBJECTIVETo explore whether angiotensin-(1-7) [Ang-(1-7)] protects cardiac myocytes against high glucose (HG)-induced injury by inhibiting ClC-3 chloride channels.

METHODH9c2 cardiac cells were exposed to 35 mmol/L glucose for 24 h to establish a cell injury model. The cells were treated with Ang-(1-7) or the inhibitor of chloride channel (NPPB) in the presence of HG for 24 h to observe the changes in HG-induced cell injury. Cell counter kit 8 (CCK-8) assay was used to test the cell viability, and the morphological changes of the apoptotic cells were detected using Hoechst 33258 staining and fluorescent microscopy. The intracellular level of reactive oxygen species (ROS) was examined by DCFH-DA staining, SOD activity in the culture medium was measured using commercial kits, and the mitochondrial membrane potential (MMP) of the cells was tested with rodamine 123 staining. The expression level of cardiac ClC-3 chloride channels was detected with Western blotting.

RESULTSExposure of H9c2 cardiac cells to 35 mmol/L glucose for 24 h markedly enhanced the expressions of cardiac ClC-3 channel protein (P<0.01). Co-treatment of the cells with 1 µmol/L Ang-(1-7) and HG for 24 h significantly attenuated HG- induced upregulation of ClC-3 channel protein expression (P<0.01). Co-treatment of the cells exposed to HG with 1 µmol/L Ang-(1-7) or 100 µmol/L NPPB for 24 h obviously ameliorated HG-induced injuries as shown by increased cell viability, enhanced SOD activity, decreased number of apoptotic cells, and reduced intracellular ROS generation and loss of MMP (P<0.01).

CONCLUSIONClC-3 channels are involved in HG-induced injury in cardiac cells. Ang-(1-7) protects cardiac cells against HG-induced injury by inhibiting ClC-3 channels.

OBJECTIVETo observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system.

METHODSA GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis.

RESULTSThe tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01].

CONCLUSIONHSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

9,10-Dimethyl-1,2-benzanthracene ; Adenocarcinoma ; chemically induced ; pathology ; therapy ; Animals ; Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Female ; Ganciclovir ; pharmacology ; Gene Transfer Techniques ; Genetic Therapy ; Herpesvirus 1, Human ; genetics ; metabolism ; Infusions, Intra-Arterial ; Ovarian Neoplasms ; chemically induced ; pathology ; therapy ; Random Allocation ; Rats ; Rats, Wistar ; Thymidine Kinase ; genetics ; metabolism

The complete gene encoding the structural protein of FMDV(VP1) was subcloned into expression vector pPROex-HT, resulting in the fusion expression plasmid pPROexHT-VP1. After transformed into E. coli BL21(DE3) and induced by IPTG, the fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. Based on the fusion protein further purified, a novel indirect ELISA (VP1-ELISA) was developed to detect FMDV antibody in pigs. Comparison between VPl-ELISA and the government standard kit (liquid phase block ELISA) showed the two methods had 96.25 percent agreement by detecting 80 serum samples, indicating that the indirect VP1-ELISA was specific and sensitive.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity ; Swine

OBJECTIVEPrader-Willi Sydrome (PWS) is a human disorder related to genomic imprinting defect on 15q11-13. It is characterized by a series of classic features such as hypotonia, hyperphagia, obesity, osteoporosis, typical facial and body dysmorphosis, hypogonadism, mental and behaviour disorders. Our study was designed to precisely detect the microdeletions, which accounts for 65%-70% of the PWS.

METHODSPhysical and laboratory examinations were firstly performed to diagnose PWS clinically, and to discover novel clinical features. Then the patient was screened with bisulfite-specific sequencing and precisely delineated through high-density array CGH.

RESULTSWith the bisulfite-specific sequencing, the detected CpG island in the PWS critical region was found homozygously hypermethylated. Then with array CGH, a 2.22 Mb type II microdeletion was detected, covering a region from MKRN3, MAGEL2, NDN, PWRN2, PWRN1, C12orf2, SNURF-SNRPN, C/D snoRNAs, to distal of UBE3A.

CONCLUSIONSArray CGH, after the fast screening of Bisulfite-specific sequencing, is a feasible and precise method to detect microdeletions in PWS patients. A novel feature of metacarpophalangeal joint rigidity was also presented, which is the first time reported in PWS.

Base Sequence ; Chromosome Deletion ; DNA Primers ; Female ; Humans ; Infant, Newborn ; Nucleic Acid Hybridization ; Prader-Willi Syndrome ; genetics