DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.
Adenosine Triphosphatases ; biosynthesis ; genetics ; isolation & purification ; Bacterial Proteins ; Base Pair Mismatch ; Chromatography, Affinity ; DNA ; metabolism ; DNA Repair ; DNA-Binding Proteins ; Escherichia coli Proteins ; biosynthesis ; genetics ; isolation & purification ; Magnesium ; pharmacology ; Molecular Weight ; MutS DNA Mismatch-Binding Protein ; Recombinant Proteins ; biosynthesis

OBJECTIVETo analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.

METHODSSix kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.

RESULTSThe reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.

CONCLUSIONThe XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.

3T3 Cells ; 5' Flanking Region ; genetics ; Animals ; Base Sequence ; Cell Line ; Chloramphenicol O-Acetyltransferase ; metabolism ; DNA ; analysis ; DNA-Binding Proteins ; genetics ; Gene Deletion ; Gene Expression Regulation ; physiology ; Genes, Reporter ; Humans ; K562 Cells ; Mice ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Promoter Regions, Genetic ; genetics ; Regulatory Factor X Transcription Factors ; Transcription Factors ; Transcription, Genetic ; physiology ; Transcriptional Activation ; Transfection ; Tumor Cells, Cultured ; X-Box Binding Protein 1

Animals ; Humans ; Leptin ; genetics ; physiology ; Liver Cirrhosis ; etiology ; RNA, Messenger ; analysis ; Receptors, Cell Surface ; analysis ; physiology ; Receptors, Leptin ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

Angiotensin II ; physiology ; Ascites ; etiology ; Humans ; Hypertension, Portal ; etiology ; Liver Cirrhosis ; etiology ; Renin-Angiotensin System ; physiology

OBJECTIVETo explore different microRNA expression profiles between chronic hepatitis B (CHB) patients of Pi-Wei dampness-heat syndrome (PWDHS) and Gan depression Pi deficiency syndrome (GDPDS).

METHODSBy applying gene chip technology, blood samples from CHB patients of PWDHS (3 cases), GDPDS (3 cases), and healthy volunteers (3 cases) were withdrawn and microRNA detected. The microRNA was screened and functional analyses performed by using SAS system.

RESULTSTotally 77 microRNAs with differential expression were screened from CHB patients of PWDHS and healthy volunteers, including 60 up-regulated microRNAs and 17 down-regulated microRNAs. Functions of target genes were mainly associated with transcription factors, gas exchange, adverse stimulating, regulation of enzyme activities, developing of the immune system, and the process of actin filaments. Totally 41 microRNAs with differential expression were screened from CHB patients of GDPDS and healthy volunteers, including 32 up-regulated microRNAs and 9 down-regulated microRNAs. Functions of target genes were mainly associated with binding to nucleotide or chromatin, inhibition and activation of transcription, biosynthesis, regulation of metabolic process, regulation of enzyme activities, developing of the immune system, the process of actin filaments, and IL-12. Totally 6 microRNAs with differential expression were screened from CHB patients of PWDHS and CHB patients of GDPDS, including 1 up-regulated microRNA and 5 down-regulated microRNAs. Functions of target genes were mainly associated with transmembrane transport, regulation of transcription factors, metabolism of hormones, developing of the immune system, the process of actin filaments, regulation of metabolic process, response to exterior stimulation, and so on.

CONCLUSIONThere existed differentially expressed microRNAs (spectrum) between CHB patients of PWDHS and CHB patients of GDPDS.

Depression ; Hepatitis B, Chronic ; genetics ; metabolism ; Hot Temperature ; Humans ; Interleukin-12 ; metabolism ; Medicine, Chinese Traditional ; MicroRNAs ; metabolism ; Oligonucleotide Array Sequence Analysis ; Research ; Syndrome

Adolescent ; Adult ; China ; epidemiology ; Disease Outbreaks ; Female ; Humans ; Incidence ; Leptospirosis ; epidemiology ; mortality ; Male ; Middle Aged

OBJECTIVETo examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.

METHODSOVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.

RESULTSThe mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).

CONCLUSIONThe data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.

Animals ; CD11c Antigen ; metabolism ; CD40 Ligand ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Liver Neoplasms ; pathology ; secondary ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; pathology ; Spleen ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

To investigate the prevalence of drug-resistance mutations, resistance to antiretroviral drugs, and the subsequent virological response to therapy in treatment-naive and antiretroviral-treated patients infected with HIV/AIDS in Henan, China, a total of 431 plasma samples were collected in Queshan county between 2003 and 2004, from patients undergoing the antiretroviral regimen Zidovudine + Didanosine + Nevirapine (Azt+Ddi+Nvp). Personal information was collected by face to face interview. Viral load and genotypic drug resistance were tested. Drug resistance mutation data were obtained by analyzing patient-derived sequences through the HIVdb Program (http://hivdb.stanford.edu). Overall, 38.5% of treatment-naive patients had undetectable plasma viral load (VL), the rate significantly increased to 61.9% in 0 to 6 months treatment patients (mean 3 months) (P＜0.005) but again significantly decrease to 38.6% in 6 to 12 months treatment patients (mean 9 months) (P＜0.001) and 40.0% in patients receiving more than 12 months treatment (mean 16 months) (P＜0.005). The prevalence of drug resistance in patients who had a detectable VL and available sequences were 7.0%, 48.6%, 70.8%, 72.3% in treatment-na(1)ve, 0 to 6 months treatment, 6 to 12 months treatment, and treatment for greater than 12 months patients, respectively. No mutation associated with resistance to Protease inhibitor (PI) was detected in this study. Nucleoside RT inhibitor (NRTI) mutations always emerged after non-nucleoside RT inhibitor (NNRTI) mutations, and were only found in patients treated for more than 6 months, with a frequency less than 5%, with the exception of mutation T215Y (12.8%, 6/47) which occurred in patients treated for more than 12 months. NNRTI mutations emerged quickly after therapy begun, and increased significantly in patients treated for more than 6 months (P＜0.005), and the most frequent mutations were K103N, V106A, Y181C, G190A. There had been optimal viral suppression in patients undergoing treatment for less than 6 months in Queshan,Henan. The drug resistance strains were highly prevalent in antiretroviral-treated patients, and increased with the continuation of therapy, with many patients encountering virological failure after 6 months therapy.

Objective Toinvestigatethestatusoftick-borneRickettsiaeinfectionsamongmurine-likeanimalsin differentareasofZhejiangprovince.Methods Liverandspleensamplesofmurine-likeanimalscapturedthroughnight trapping method were collected from Anji,Jinhua and Tiantai County according to their geographic locations and historical detection of Rickettsiae .Nest-PCR tests were used to determine the presence of the 16S rRNA genes of Anaplasma and Ehrlichia ,and the heat shock protein genes (groEL)of Rickettsiae (including typhus and spotted fever group)and Orientiainthesesamples.Results Atotalof851murine-likeanimalsbelongingto14specieswerecaptured.The predominant species were Rattus confucianus (30.32%),Apodemus agrarius (18.80%) and Thallomys paedulcus (1 1.75%)and they were significantly different among three areas (P<0.05 ).48 Rickettsia positive were found in 562 tested samples with the positive rate of 8.54%,among which the percentage of Anaplasma,typhus group Rickettsia, Orientia,Ehrlichia and spotted fever group Rickettsia were 3.38%,1.78%,1.78%,1.07% and 0.53% respectively. The positive rates of Anaplasma in Jindong (4.76%)and Anji (4.27%)were significantly higher than that in Tiantai (P<0.05 )while the spotted fever group Rickettsia were found only in Tiantai County.Moreover,Rattus confucianus-the predominant species of Zhejiang Province-had the highest infection rate of tick-borne Rickettsiae up to 14.97%.Co-infections with several Rickettsiae were existed among the same species.Conclusion Rickettsiae infections exist widely among different areas of Zhejiang province and the positive rates are significantly different among species.

BACKGROUND/OBJECTIVES: Increased levels of uremic toxins and decreased antioxidant capacity have a significant impact on the progression of chronic kidney disease (CKD). However, it remains unclear whether they interact with each other to mediate the damage of kidney function. The purpose of this study was to investigate whether uremic toxins (i.e., homocysteine and indoxyl sulfate [IS]), as well as glutathione-dependent antioxidant enzyme activities are dependently or independently associated with kidney function during different stages of CKD patients. SUBJECTS/METHODS: One hundred thirty-two patients diagnosed with CKD at stages 1 to 5 participated in this cross-sectional study. RESULTS: Patients who had reached an advanced CKD stage experienced an increase in plasma uremic toxin levels, along with decreased glutathione peroxidase (GSH-Px) activity.Plasma homocysteine, cysteine, and IS concentrations were all positively associated with each other, but negatively correlated to GSH-Px activity levels after adjusting for potential confounders in all CKD patients. Although plasma homocysteine, cysteine, IS, and GSHPx levels were significantly associated with kidney function, only plasma IS levels still had a significant association with kidney function after these parameters were simultaneously adjusted. In addition, plasma IS could interact with GSH-Px activity to be associated with kidney function. CONCLUSIONS IS plays a more dominant role than homocysteine and GSH-Px activity in relation to kidney function.