Objective:Ghrelin is a brain-gut peptide with GH-releasing,apetide-inducing and anti-inflammation activities and with widespread tissue distribution.Ghrelin is the endogenous ligand of GH secretagogue receptor (GHSR),and both ghrelin and the GHSR are expressed in T cells.We therefore examined the effect of Ghrelin on human T cell and its signal transduction.Methods:Ghrelin-activating mTOR pathway in human primary T cell was studied using immunoblotting and inhibitors of the PI3K(LY294002,3-Methyladenine) or mTOR(rapamycin) and antagonist of GHSR1a(Des-Lys-3-GHRP6).Results:The results showed that GHSR1a was expressed on T cells.Ghrelin caused a significant increase in the phosphorylated mTOR,P70S6K,S6K,4E-BP-1,eIF4G,eIF4E by immunoblotting.While the phosphorylated mTOR,P70S6K were abolished by the mTOR inhibitor rapamycin and PI3K inhibitor LY294002,3-methyladenine and also antagonist of GHSR1a,Des-Lys-3-GHRP6.Conclusion:The data document that Ghrelin activates translation of T cells through mTOR pathway.

Objective To investigate clinical significance of plasma D-dimer (D-D),fibrinogen (FIB) and fibrin/fibrinogen degradation product (FDP) in patients with chronic obstructive pulmonary disease (COPD).Methods The level of plasma D-D,FIB and FDP in 150 patients with COPD and 80 healthy persons were detected,and compared.Results The level of plasma D-D,FIB and FDP in COPD patients were significantly higher than those in healthy persons[(2.16 ± 0.61) mg/L vs.(0.55 ± 0.04) mg/L,(5.88 ± 1.52) g/L vs.(3.12 ± 0.35) g/L,(7.18 ± 1.63) mg/L vs.(3.62 ± 1.55) mg/L],there were significant differences (P ＜ 0.01).Conclusion Monitoring the level of plasma D-D,FIB and FDP in COPD patients can provide reliable basis in hypercoagulable state and primary and secondary hyperfibrinolysis.

Objective To investigate the linkage between the mutation of IL 4R ? chain gene and susceptibility of asthma and atopy in Wuhan. Methods Thirty five samples from asthmatic children,twenty five samples from adult patients with asthma, twenty samples from health adult and children respectively, were analyzed. A coding region 1205 1928 of IL 4R ? chain gene was detected by reverse transcription polymerase chain reaction/Single Strand conformation polymorphism/Sequence. Result 57.8% cases from asthmatic children, one polymorphism (1902 A→G), were found exist in the IL 4R ? chain gene and only 8% cases from asthmatic adults and 5% cases from control have this mutation. Conclusion The SNP of IL 4R ? chain coding area 1902 correlates with susceptibility of asthma in Wuhan asthmatic children (? 2=12.54, P 0.05, ?=0.05). There may be another mutations in IL 4R ? chain coding area except 1902 or another changes in other gene related with allergy.

Objective The aim of this study was to establish a new method for genotyping ATP7B Arg778Leu gene mutation that does not require RFLP PCR or sequencing.Method 4-primer amplification refractory mutation system (ARMS)-PCR was performed to screen the Arg778Leu mutation in 47 unrelated Wilson's disease (WD) patients and 30 unrelated healthy controls.Direct sequencing was used to confirm the specific amplification products.Results PCR products were visualized on agarose gel electrophoresis.Among the 47 WD patients,4 were homozygous and 14 were heterozygous for this mutation.The total mutation rate was 38.3% (18/47).The results of direct sequencing completely consisted with the results of 4-primer ARMS-PCR.Conclusions The ATP7B Arg778Leu gene mutation is a hot spot for the research of Chinese WD patients.4-primer ARMS-PCR is a fast convenient and accurate method for typing mutation in high throughput population screening.This approach can be used to detect other point mutations.

Outwardly rectifying swelling-activated chloride conductance (ICl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, ClC-4 and ClC-5) were determined using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4' isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of I(Cl,Swell), including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.
Biophysics/methods ; Chlorides/*chemistry ; Chlorides/metabolism ; DNA Primers/chemistry ; Electrophysiology/methods ; Gene Expression Regulation ; Glycolates/pharmacology ; Heart Ventricles/*cytology ; Ischemic Preconditioning ; Muscle Cells/*cytology ; Osmosis ; Sequence Analysis, DNA

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown etiology , a median survival time of which is 2 to 3 years.The diagnosis and treatment are important for IPF in time.Krebs von den lungen-6(KL-6), Surfactant protein-A(SP-A) and Surfactant protein-D(SP-D) are acceptable biomarkers in clinical for idiopathic pulmonary fibrosis in Japan,which have shown good sensitivity at diagnosis IPF and predict the prognoses for patients with IPF . However , the differential diagnosis of IPF from other interstitial lung diseases is still challenging .Other biomarkers are being developed , one of which would have the best specificity and sensitivity at diagnosis IPF.Those biomarkers about pathogenesis of IPF includes alveolar epithelial cell dysfunction , fibrogenesis and immune dysregulation are shown .They are potential to account for underlying disease mechanisms , accelerated drug development and advance clinical management.

Outwardly rectifying swelling-activated chloride conductance (lCl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myoeytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, CIC-4 and CIC-5) were deter- mined using RT-PCR and Western blot analysis.Whole cell ICl,Swell was recorded from isolated rabbit ventricular myoeytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4′ isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was con- firmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of ICl,Swell including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.

Objective To explore the peptides that can mimic the blood type A antigen and evaluate the anti-A antibody detection value of these peptides.Methods The anti-A monoclonal antibodv (NaM87-1F6)was used to panning the phage clones from a phage display 12-mer peptide library.Positive clones were identified by phage ELISA,phage mieropanning methods.Phage DNA Was sequenced and the corresponding peptide sequences were deduced.Agglutination inhibition test WaS performed to assess the ability of phage clones to inhibit the binding between the type A red blood cell and the anti-A antibody. ABO-ELISA based on the selected peptides was compared with classical haemagglutination test jn the detection of senlm anti-A antibody.Results Seven positive clones were chosen after panning,phage ELISA and phage micropanning.Six clones displayed peptide EYWYCGMNRTGC(C5),the other one displayed peptide QIWYERTLPFTF(C17).The phages displaying the selected peptides could specifically inhibit agglutination of type A red blood cells(RBCs)by anti-A antibodies.In the ABO-ELISA based on C5 and C17,the receiver operating characteristic(ROC)Curve showed that area under curve(AUC)were 0.889 (P=0.000),0.75l(P=0.000)respectively.The Spearman correlation Coeffieient between the ABO-EliSA value and the antibody titer derived from haemagglutination assay were 0.743(P<0.01),0.664(P<0.01)respectively.As for C5,0.300 was the best cut-off for ABO-ELISA with 82.2% sensitivity and 83.3% specificity.As for C17,the sensitivity and specificity of ABO-ELISA was 68.9% and 63.3% respectively when the cut-off value was 0.250.Conclusions The peptides EYWYCGMNRTGC and QIWYERTLPFTF can mimic the blood type A antigenic epitope.ABO-ELISA based on these peptides has the potential for the detection of anti-A antibody.

Objective To understand the expression of mRNA for two Toll-like receptors (TLRs) (TLR2 and TLR4) and two T helper (Th)1 and Th2 cytokines [ interleukin (IL)-4, interferon (IFN)-gamma] in splenic macrophages of ovalbumin-induced allergic asthma mice model. Methods The expression of mRNA for TLB2、TLR4 and the house-keeping gene HPRT in both splenic macrophages of BALB/c allergic asthma mice and the control mice were analysed by real-time polymerase chain reaction (PCR). We examined the threshold cycle times (Ct) of cDNA of TLR2、TLR4 and HPRT gene to get Ct2、Ct4 and CtH respectively. The value of relative quantity of mRNA level was calculated by the ratio of Ct2/ CtH、Ct4/CtH. After stimulating cells with PMA and Ionomycin with anti-CD3/CD8 in the presence of Brefeldin A, intracellular levels of Th1 cytokines (IFN-?) and Th2 cytokines (IL-4) were determined for CD3+ CD8- and CD3+ CD8+ T cells. Results We observed Ct2/CtH (1. 26?0. 04,1. 35?0. 11, P 0.05) in splenic macrophages between the control mice and allergic asthma mice; the expression of TLR2 mRNA of allergic asthma mice was significantly reduced. We examined IFN-?[IFN-?(20. 83?1. 32)%, (31.78?2. 54) %, IL4(2. 04?0. 24)% , (5. 91?0.83)% P

Aim To study the effects of dexamethasone(DXM)and peroxisome-proliferation activated receptor?(PPAR?)agonist pioglitazone on intracellular cytokines expression of T_H1/T_H2 and the mechanism of that in asthma model,and compare the difference between two drugs.Methods 24 Balb/c mice were divided into 4 groups randomly: control group,asthma group,DXM group,and pioglitazone group,with 6 mice in each group.The expression of T-bet and GATA3 of lung were detected using western blot,and the intracellular cytokines interluekin 4 and interferon ? expressions of CD~(4+)Tcell were measured using flowcytometry.Results Compared with control group,the level of the expression of T-bet of lung in asthma group increased significantly(P0.05),while both of GATA3 and T-bet decreased after DXM treatment,and the decreasing degree of GATA3 was more than that of T-bet.The results of flowcytometry indicated that the ratio of intracellular cytokines IL-4/IFN? of CD~(4+) T cell in asthma group was much higher than that of control group(P