Objective To investigate the relationship between protein phosphatase inhibition, tau hyperphosphorylation and neuronal death seen in Alzheimer disease (AD). Methods Co culture of protein phosphatase inhibitor okadaic acid (OA) and neuroblastoma cells (SH SY5Y), by agarose gel electrophoresis to detect DNA fragmentation, and in situ hybridization by TdT mediated biotin labeled dNTP nick end labeling (TUNEL) to further detect the cell apoptosis. Results Incubation of SY5Y cells with 10 nmol/L OA for 24 or 48 hours led to the appearance of DNA fragmentation and a remarkable increase of positive cells from 2 16%?0 94% to 18 05%?3 57% ( P

BACKGROUND: Central nervous system (CNS) myelin is made up of 70% lipids and 30% proteins with human brain myelin basic protein (hMBP) constituting 1/3 of the proteins. MBP is a family of proteinswith four isoforms only in human brain. OBJECTIVE: To investigate the expression of MBP and its immunological function. DESIGN: Single sample study. SETTING: Department of Biochemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University. MATERIALS: This experiment was conducted at Department of Bio chemistry and Molecule Biology, Huaxi College of Priclinical Medicine and Forensic Medicine, Sichuan University from August 2003 to March 2004. Relative molecular weight was 215000 hMBP cDNA clone pGEMP,prokaryotic expression recombinant vector pGEX-5T, T4 DNA Ligase,calf intestine alkaline phosphates, X-gal, IPTG, 123 Ladder (BRL), nitrocellu lose ,4-chloro-1-Naphthol;MBP and Anti- MBP ELISA kit. INTERVENTIONS: ①hBMP gene cDNA clone segment was digested with EcoR1 and BamH1; ②Construction and transformation of the recombinant expression vector p5TMP; ③ The growth and induced expression of the recombinant expression vector transformed bacteria; ④ Preparation of the antibody of recombinant hMBP. MAIN OUTCOME MEASURES: ① Detection and identification of the expressed protein product; ② Detection and identification of hMBP anti body. RESULTS: ① Screening and identification of recombinant MBP: The 4 999 bp pGEX-5T DNA and 557 bp MBP inserted fragment were obtained, indicating that the white colonies contained recombinant expression plasmid of exogenous DNA; ② A dense band disappeared from the control plasmid while a new special polypeptide band with apparent molecular weight 42 000 was detected in recombinant cell lysate; ③After 5 subcutaneous injections, antibody was obtained at 1:16 titer. The specificity of the antibody to MBP was confirmed.CONCLUSION: Compared with the original expression vector, the new constructed expression vector containing 215 000 MBP exons Ⅰ-Ⅶ coding sequence was not only without the coding area defects of 34 bp in 5' sequence but also expressed more highly in prokaryote obviously. In addition, the recombinant MBP antibody prepared successfully.

Objectives: To assess the feasibility and stability of right ventricular outflow tract (ROVT) pacing under current technology by comparing the results of ROVT pacing with the traditional right ventricular apex (RVA) pacing. Methods: A total of 42 patients (at mean age of 63.5±10.4 years) without structural heart disease were randomly divided into two groups. RVA pacing group (n=14),and RVOT pacing group(n=28). An active fixation lead was implanted in all patients whose pacemaker could automatically measure the pacing threshold every day. The operation time,X-ray exposure time and lead parameters detected during the operation were collected to evaluate the feasibility of RVOT pacing. The complications related to lead and implantation procedure and the trend of threshold change during the follow-up time were used to assess the stability of RVOT pacing.Results: There were no statistic differences between RVA pacing group and RVOT pacing group in terms of operation time,X-ray exposure time and lead parameters. In RVOT group,the change of threshold during acute period was similar to those in RVA group (P=0.23). Chronic pacing threshold was also comparable between two groups,mean threshold at 6 months follow-up time was 0.55±0.11V and 0.54±0.09V at 0.4 pulse width in RVA group and RVOT group respectively (P=0.787).Conclusion: RVOT pacing was feasible and stable in operation time and lead characteristics compared with the conventional RVA pacing under current pacing technology.

The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.

OBJECTIVEThe purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy.

METHODSThe 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E. coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method.

RESULTSThe sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other.

CONCLUSIONThe authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.

Animals ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genes ; genetics ; Genes, MHC Class I ; genetics ; Genetic Therapy ; H-2 Antigens ; genetics ; Mice ; Mice, Inbred C57BL ; Point Mutation ; Sequence Analysis, DNA ; Transgenes

Objective To evaluate the copy number variation of FCGR3B gene in Henan Han systemic lupus erythematosus (SLE) patients and healthy controls,and explore the association between FCGR3B gene copy number variants (CNVs) and lupus nephritis (LN) susceptibility in Henan Han population.Methods FCGR3B CNVs was investigated in 142 SLE patients with nephritis,187 SLE patients without nephritis and 328 healthy controls.A modified methodology based on competitive PCR named Multiplex AccuCopyTM Kit was used to detect FCGR3B copy number.Clinical and laboratory data were collected retrospectively from the medical record.Logistic regression analysis was used to determine the association of FCGR3B copy number variants with LN susceptibility.Rank correlation was used to determine the correlations between FCGE3B copy number variants and clinical phenotypes of LN.Results No significant difference was detected in the copy number variations of FCGR3B in different groups.Low copy number of FCGR3B was more commonly seen in patients with nephritis (P=0.042),and was a risk factor for LN (OR=2.059; 95％ CI:1.081-3.921; P=0.028).However,high copy number (＞ 2) had no effect on SLE patients without nephritis(OR=1.152; 95％CI:0.711-1.866; P=0.565) and LN patients (OR=0.838; 95％ CI:0.529-1.329; P=0.454).There were no associations between FCGR3B copy number variants and clinical phenotypes and immunologic characteristics of LN.Conclusion The low copy number of FCGR3B is a risk factor for LN in Henan Han population.

We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector. The recombinant vector p5TMP was transformed into E. coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning. Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1:16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.
Animals ; Antibody Formation ; Autoantibodies ; Brain ; immunology ; metabolism ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Myelin Basic Protein ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology

Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.

Objective:To investigate the effects of oxidized low density lipoprotein (Ox-LDL) on cell proliferation and mRNA expression of inflammatory factors in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).Methods:Tissue culture was used to isolate and 4-6 generation cultured RA-FLS cells were used for subsequent experiments. RA-FLS were stimulated for 24 hours with different con-centr-ations of human Ox-LDL, then the MTS cell proliferation and toxicity test kit were used to detect the prolifer-ation of RA-FLS. Real time-polymerase chain reaction (RT-PCR) was used to test the expression of inflamm- atory factors like interleukin (IL)-6, transforming growth factor (TGF)-β, IL-8, tumor necrosis factor (TNF)-α and receptors like CD36 and scavenger receptor binds phosphatidylsed neoxidized lipoprotein (SR-PSOX) inRA-FLS. T test and F test were used in this study. Results:Ox-LDL (10, 25, 50 μg/ml) could obviously promote the proliferation of RA-FLS, and theabsorbance values (490 nm) were (1.04±0.15), (1.05±0.14), and (1.00±0.10), respectively, all higher than the control group (0.81±0.04) and the difference was statistically significant ( F=4.737, P<0.01). In addition, 50 μg/ml and 100 μg/ml Ox-LDL also promoted the expression of IL-6 mRNA ( F=14.709, P<0.01) and inhi-bited the expression of TGF-β mRNA ( F=299.074, P<0.01), but there was no obvious effect on the expression of IL-8 and TNF-α. Ox-LDL stimulation could obviously promote the expression of SR-PSOX receptor on RA-FLS ( F=68.636, P<0.01) and inhibit the expression of CD36( F=18.085, P<0.01). After the transfection of siRNA, SR-PSOX mRNA level was significantly inhibited and the mRNA expression of IL-6 was significantly decreased after Ox-LDL stimulation of RA-FLS ( t=3.875, P<0.01), while TGF-β mRNA expres-sion was not significantly changed( t=-0.193, P>0.05). Conclusion:Ox-LDL may play a role in promoting the activation of RA-FLS proliferation and the expression of IL-6 mRNA by increasing the SR-PSOX receptor of RA-FLS, suggesting that Ox-LDL is involved in the synovial inflammation of RA.

Objective To evaluate the effect of hypernatremia in donors on perioperative recovery of liver function in the recipients undergoing liver transplantation. Methods Clinical data of 73 liver transplant recipients were analyzed retrospectively. According to the serum levels of sodium in donors, all recipients were divided into hypernatremia group (donor serum sodium ≥150 mmol/L, n=19) and non-hypernatremia group (donor serum sodium < 150 mmol/L, n=54). Serum alanine aminotransferase(ALT), aspartate aminotransferase (AST), model for end-stage liver disease (MELD) score, albumin, total bilirubin (TB), serum creatinine, prothrombin time and hepatocyte growth factor (HGF) in the recipients were detected at 1, 3, 7, 14 and 21 d after liver transplantation. The time of postoperative use of liver-protecting drugs in the recipients, the length of intensive care unit (ICU) stay, the average length of hospital stay and the incidence rate of postoperative complications were statistically compared and analyzed. Results Compared with the non-hypernatremia group, the serum levels of TB, ALT, AST, HGF and MELD scores of the recipients in the hypernatremia group at the postoperative 1, 3 and 7 d were significantly higher (all P < 0.05), whereas the serum albumin level was significantly decreased (P < 0.05). The prothrombin time in the hypernatremia group was significantly longer than that in the non-hypernatremia group at 3 and 7 d after operation (both P < 0.05). In the hypernatremia group, the time of postoperative use of liver-protecting drugs and the length of ICU stay were 9 (7-13) d and 11 (8-13) d, significantly longer than 4 (3-9) d and 7 (3-9) d in the non-hypernatremia group (both P < 0.05). The average length of hospital stay, serum creatinine level and incidence rate of postoperative complications did not significantly differ between two groups (all P>0.05). All recipients were recovered and discharged. Conclusions The hypernatremia in donors exert no significant effect on the perioperative liver function of the recipients, whereas it can prolong the postoperative recovery time of liver function of the recipients.