AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-? (TNF-?)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-? or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-? (10~5 (U/L)) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 ?mol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 ?mol/L), the specific sGC inhibitor, and Chel (5 ?mol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 ?mol/L), ODQ, Chel, antoxidant 2-MPG (400 ?mol/L) or tyrosine kinase inhibitor genistein (50 ?mol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-?. CONCLUSION: The results suggest that NO may play a role in TNF-?-induced cardioprotection, which is mediated by sGC and PKC. [

Objective To set up and verify a new scoring system of clock drawing test(CDT).Methods CDT and other neuropsychological tests were applied to 180 normal individuals.170 subjects with mild cognitive impairment(MCI),and 31 patients with mild Alzheimer's disease(AD).The first step of the 30-score system of CDT was to anchor the 4 points of"12-3-6-9".totaling a score of 4 which was the "30-score system A(Anchoring)part";and the other 13 items reflecting the drawing results and totaling a score of 26 made up the so called"30-score system C(Clockfaee)part".the two together had a total score of 30.Thirty-eight subjects underwent Xe-enhanced computed tomography(Xe-CT)scanning for quantitively measuring regional cerebral blood flow(rCBF)of 26 regions of brain.Results Correlations coefficients of the scores of 14 items with the total score in 30-score system of CDT were in the range of 0.48to 0.71(P＜0.01).30-score system C part was closely correlated with indicators of visuospatial and executive function,while 30-score system A part with memory indicators.The sensitivity and specificity of the 30-score system A part score≤2 for detecting MCl were 70.6%and 73.9%.respectively.The sensitivity the specificity of 30-score system C part score≤17 for mild AD were 75.3%and 75.9%.respectively.In the regression equation of 30-score system A and C part.the combined independent variables were the CBF of left frontal cortex and right white matter,respectively.Conclusion The 30-score system A and C part have difierent meanings:30-score system C part could be used to identify AD while 30-score system A part is helpful for identiring MCI.

AIM: To clone and express a human monoclonal anti-D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobulin molecules of anti-D. METHODS: The gene of anti-D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS-PAGE and ELISA. RESULTS: The result of SDS-PAGE confirmed that E.coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supernatant reacted with Rh+ group O human erythrocytes, but was not recognized by Rh-group O human erythrocytes. CONCLUSION: Expressed Fab fragment has the antigenic specificity for human erythrocytes.

AIM: To amplify from leader peptide region an d obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.ME THODS: The total RNA was extracted from an Epstein-Barr-virus-transforme d cell line secreting monoclonal anti- (rhesus D) antibody. The leader region pri mers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM ?) variable region gene was amp lified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previ ously published sequences.RESULTS: A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primer s, respectively. Sequence analysis indicated that the deduced amino acid sequenc e w as in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION: The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for pro duction of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns.

Objective:To differentiate human embryonic stem cells ( hESCs ) into keratinocytes ( K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs.Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium.The hESCs were directly differentiated into kerati-nocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 sup-plement.The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Real-time PCR.Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques.Results:H9-hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium.The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT ( P<0.05) , but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs ( P >0.05 ) .Conclusion: H9-hESCs could be directly differentiated into K-hESCs.The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.

Objective To evaluate the diagnostic value of fluorescent quantitative polymerase chain reaction(PCR) for Mycoplasma pneumoniae (MP) in children with MP pneumonia(MPP).Methods From Jun.2008 to Jan.2009,153 cases hospitalized with pneumonia were enrolled,and 30 cases without respiratory infection were enrolled as control group.Their respiratory secretion (including nasopharyngeal secretion,sputum,bronchialalveolar lavage fluid or pharyngeal swab) samples were collected for fluorescent quantitative PCR for MP.And their single or paired serums were collected for specific MP antibody detection.Results There were 123 cases confirmed with MPP by serology,among whom 114 cases were MP PCR positive.The quantitation of MP DNA was among 1.20?106-3.66?1010 gene copys/L. There were 30 cases with pneumonia negative with MP by the paired serum serology,among whom 2 cases were MP PCR positive,and the quantitation of MP DNA was (1.08-3.02)?107gene copys/L.All cases of control group were MP PCR negative.During the first and second weeks of the MPP onset,the sensitivity of MP-IgM from the first single blood samples were 66.7% and 83.9%,respectively.While the sensitivity and specificity of MP PCR were 92.7% and 93.3%,respectively.From the third week of the disease onset,the sensitivity of MP-IgM from the first single blood samples increased to 90.9%-100%.The clinical manifestations of MPP were nonspecific.Conclusions PCR is superior to serology for early diagnosis on MP infection.Combination of the 2 methods may be helpful to early and accurate diagnosis on MP infection.

Placentation,which is critical for maternal-fetal exchange of nutrients and gases,is a complicated process comprising stepwise vasculogenesis and angiogenesis.Hypoxia caused by impairedtrophoblast invasion may cause various angiogenic abnormalities in human placenta.The Notchl signaling pathway plays an important role in the regulation of angiogenesis.The angiogenesis of human umbilical vein endothelial cells (HUVECs) under normal/hypoxic conditions and the mRNA/protein level of Notchl/Dell4/Jaggedl were investigated in this study.The effects of DAPT/JAG-1 on the migration of HUVECs were also assessed by cell wound healing assay,so as to discover the possible role of notchl signaling pathway in the angiogenesis of human placenta.The results showed that angiogenic ability of HUVECs was seriously reduced under hypoxic conditions.The mRNA and protein levels of Notchl/Dell4/Jaggedl were decreased in the hypoxic group compared to the control one.In addition,the migration capability of HUVECs was significantly obstructed when treated with DAPT and under hopoxic condition,but promoted when treated with JAG-1.The above results demonstrate that hypoxia downregulates the angiogenesis in human placenta via Notch 1 signaling pathway.

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection

Objective To clone and express the thioredoxin(Trx)from RH strain tachyzoites of Toxoplasma gondii,estab?lish the prokaryotic expression vector and purify the recombinant protein,then produce the polyclonal anti?Trx antibody in rab?bits. Methods Trx fragment was amplified by PCR and cloned into the pET?28a(+)vector,and the recombinant protein was in?duced with IPTG and purified by Ni?NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET?28a(+)was use?fully constructed,and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also ob?tained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti?Trx antibody,which will facilitate the biological functions of Trx.

This study aimed to analyze the etiology of the encephalitis outbreak in Longyan, Fujian Province, China in 2010, in order to provide valuable information for this prevention and control of this disease. Pathogens were confirmed from cerebrospinal fluid samples with fluorescent RT-PCR, virus isolation (RD cells), and neutralization tests. Then, the VP1 fragments or whole genome nucleotide sequences were determined for four virus strains using PCR. Homology was assessed using the MegAlign software, and a phylogenetic evolutionary tree was drawn using Mega 4.0 software. The results confirmed that the etiology of the outbreak was the ECHO6 intestinal virus, and the nucleotide sequence of the VP1 segment indicated that the C2 subtype was responsible. The genome sequence consisted of 7407 nucleotides, and resembled the genome of other ECHO and CoxB viruses with homology levels of 78.5%-87.3%. The encephalitis outbreak in Longyan in 2010 was caused by the ECHO6 C2 subtype intestinal virus, and its complete genome sequence length is similar to the standard strain (U16283) with a sequence homology of 80.4%.
Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Echovirus 6, Human ; classification ; genetics ; isolation & purification ; Echovirus Infections ; epidemiology ; virology ; Encephalitis ; epidemiology ; virology ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny