Objective To investigate the molecular mechanism of translocation of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1 )to the cytoplasm after glucose starvation and the effects of increased cytoplasmic translocation of hnRNPA2B1 on the survival of prostate cancer PC3 cells. Methods Human prostate cancer PC3 cells were divided into normal control group (cultured conventionally with glucose-containing medium,RPMI 1640 Medium)and glucose starvation group (cultured with glucose-free medium,RPMI 1640 Medium).The 2 types of cells were treated with deacetylase inhibitor,trichostatin A (TSA ) combined with nicotinamide (NAM),AKT inhibitor BEZ235,si-NC transfection,and si-hnRNPA2B1 transfection,respectively.Cytoplasmic and nuclear protein separation,immunoprecipitation and Western blotting were used to detect changes in hnRNPA2B1 acetylation,total AKT protein and its phosphorylation level,and expression levels of hnRNPA2B1 in the cytoplasm and nucleus.CCK-8 assay was employed to observe cell survival in each group.Results After 3～5 h of glucose starvation treatment,the acetylation of hnRNPA2B1 protein was reduced (P<0.01 ),and its cytoplasmic translocation was increased in PC3 cells (P<0.01 ),which was accompanied by enhanced AKT phosphorylation and activation of the AKT signaling pathway.TSA/NAM treatment,BEZ235 treatment,and si-hnRNPA2B1 transfection all resulted in obvious increase in acetylation of hnRNPA2B1 protein when compared with glucose starvation treated cells (P<0.01 ),which could inhibit the glucose starvation-mediated cytoplasmic translocation of hnRNPA2B1,suppress AKT phosphorylation,and consequently decrease the cell survival rate after glucose starvation (P<0.01).Conclusion Glucose starvation can maintain the survival of PC3 cells by inducing the activation of the Ac-hnRNPA2B1-AKT signaling pathway.

Objective To explore the mechanism,clinic features and treatment of type Ⅳ cardiorenal syndrome.Methods The clinical data of one patient with cardiorenal syndrome characterized with chest distress was analyzed.Results After combination treatment,the symptoms were relieved,the amount of physical activity was increased,and the functions of heart and kidney were improved.Conclusions Active,prompt and rational multidisciplinary care can control the progression of cardiorenal syndrome,increase survival rate and improve life quality.

Designed experiment is a new experimental mode characterized by the student-centered and toacher-oriented teaching principle.To conform with the general trend of experimental teaching reform in medica university,Biochemistry Department of Third Military Medical University attemptod to adopt this teaching mode.It was indicated that designed experiment is favorable for the enhancement of comprehensive diathesis and will be an effective way to cultivate high-quality medical intellectuals.

Through introducing and summarizing the application of multimedia technology in the process of biochemistry teaching,the article discusses the contribution of multimedia technology in teaching method,teaching organization,teaching content and so on and points out the importance of multimedia technology in biochemistry teaching.

Objective To construct an expression vector pET32a(+)/SLC and express the fusion protein TrxA SLC. Methods Total RNA from human inflammatory tonsil was extracted and its cDNA was generated with reverse transcription. Mature secondary lymphoid tissue chemokine (SLC) gene was amplified by RT PCR and cloned into plasmid pET32a(+) with Nco Ⅰ and Eco RⅠ sites added to the 5′ and 3′ ends respectively. E. coli DH5? was transformed with the recombinant plasmid, and the grown clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. The 3 amino acids between enterokinase site and target gene were deleted with mutation and the new vector was verified by sequencing. Expression of SLC was analysed by SDS PAGE. The fusion protein was purified by metal affinity chromatography and weak cation exchange chromatography, which was analysed by SDS PAGE and Western blotting. Results Trx SLC fusion protein expression vector was successfully construced, and the fusion protein was expressed with solubility. The purified fusion protein displayed the ability of binding the goat anti human SLC polyclonal antibody. Conclusion The SLC fusion protein can be expressed with stability and solubility and primary purification is performed with metal affinity chromatography and weak cation exchange chromatography.

Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5? system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.

Objective To clone the cDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into vector pUC19. After sequence analysis, the cDNA was ligated into prokaryotic expression vector pQE-80L and induced by IPTG to express HMGB1. The protein was purified with Ni~(2+)-NTA chromatography and polymyxin B affinity column. To identify the function of purified protein, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pQE-80L/HMGB1 was constructed successfully. After purification, the protein purity reached 96%. The recombinant HMGB1 stimulated THP1 to secrete TNF-? . Conclusion The highly purified HMGB1 was obtained successfully, which showed biological activity. These results lay the foundation for further research on the function of human HMGB1.

OBJECTIVETo generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.

METHODSBalb/c mice were immunized i.p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.

RESULTSThe VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed beta or gamma type anti-Id ScFv.

CONCLUSIONThe anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; genetics ; Antibodies, Monoclonal ; genetics ; immunology ; Bacteriophages ; genetics ; Cloning, Molecular ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin Light Chains ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; Stomach Neoplasms ; immunology ; Vaccines, DNA ; genetics ; immunology

Objective To observe the pathological changes of liver cells in the early periods of burn injury combined with endotoxemia and to explore its mechanisms preliminarily. Methods Rats were inflicted with 20% TBSA Ⅲ degree burn combined with intraperitoneal injection of lipopolysaccharide (burn injury combined with endotoxemia, combined group) or simply burned or simply injected. Each group included 25 rats, with 5 rats left for control. Rats in the former 3 groups were sacrificed and sampled at 1, 3, 6, 12 and 24 h after injury, 5 rats for each time point. The results were observed by HE staining, TUNEL and DNA gel electrophoresis, and the concentrations of plasma MDA and the activity of plasma SOD were assessed and analyzed. Results Apoptosis of liver cells was found in each of the 3 groups but its regularity was different from one another. Much more apoptotic cells in the combined group were found than the other 2 groups and apoptosis happened earlier with its maximum at 6 h postinjury and declined thereafter. Much less apoptotic cells were found in the simply burned group and reached the maximum at 12 h postburn. There were only a few apoptotic cells at 6 h postinjection in the simply injected group in which there were the fewest apoptotic cells in the three groups. The concentration of plasma MDA and the activity of SOD always moved in the opposite way at the same time. In the combined group, the maximum of MDA concentration and the minimum of SOD activity happened at the same time point, which was just before the one of the maximum of liver cell apoptosis. Conclusion During the early period of burn injury combined with endotoxemia, rat liver cells die mainly in the apoptotic way, with lots of apoptotic cells at 6 h and 12 h postinjury, combined with necrosis at 24 h. Lipid peroxidation may partly account for the apoptosis of liver cells.

Objective:To generate phage displayed anti idiotypic antibody single chain variable fragments(anti Id ScFv)to monoclonal antibody MC3 directed against colorectal carcinoma.Methods:Balb/c mice were immunized i.p. with MC3(McAb against colorectal Carcinoma) conjugated with KLH,and mRNA was isolated from the spleens of the immunized mice.VH and VL DNAs of the antibody were amplified separately and assembled into ScFv DNAs with a linker DNA by PCR.The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield phage antibody ScFv library.After four rounds of panning to the library with MC3,the MC3 positive clones were selected by ELISA from the preselected phages.Results:The VH,VL and ScFv DNAs were about 340,320 and 750 bp respectively.After four rounds of panning to the antibody library,15 MC3 positive phage clones displayed anti Id ScFv were selected from 50 enriched phage clones.Conclusion:The phage displayed anti Id ScFv to MC3 were successfully selected by phage antibody library technology,which might provide new putative candidate molecules for developing recombinant anti Id vaccine of colorectal carcinoma.