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.

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.

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

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.

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 verify the performance of quantitative detection of interleukin-6 by using IMMUNITE1000 chemilumi-nescence analyzer.Methods According to the requirements of International Organization for Standardization(ISO)1 5 189,serum specimen were collected and levels of IL-6 were detected.The precision,accuracy,analytical measurement range,reportable range amd normal reference range of quantitative detection of interleukin-6 by using IMMUNITE1000 chemiluminescence analyzer were verified,and its performance was evaluated.Results The coefficient variation(CV)of between-day precision of high and low value was 6.42% and 1.97% respectively,and that of within-run precision was 3.40% and 3.82% respectively.Compared the test re-sults with the target values,the bias % was 0.91%.The regression equation:Y =0.986X - 7.1 (r 2 = 0.999,P < 0.05 ).With 27 times diluted,the recovery rate was from 97% to 100%,and the clinical reportable range was 2 to 27 000 pg/mL.The 95% refer-ence interval ranged from 0 to 5.3 pg/mL.Conclusion The performance of this system meets the manufacturer′s declaration,and could satisfy the quality requirements of clinical laboratory.

Autoimmune blistering skin diseases are a group of organ-specific autoimmune disorders that are characterized by autoantibodies against desmosome and hemidesmosome which are structural proteins of the epidermis or the dermal-epidermal junction and clinically by blisters and erosions on skin and/or mucous membranes.According to the skin level at which the blister occurs and the structural proteins that the autoantibodies target,autoimmune blistering diseases can be categorized into intraepithelial blister group and subepidermal blister group.The treatment options and prognosis are different among the various diseases.Since clinical criteria and histopathological characteristics are not sufficient for an accurate diagnosis of autoimmune blistering skin diseases,direct immunofluorescence microscopy,indirect immunofluorescence microscopy,ELISA,immunoblotting and immunoprecipitation are needed for exact diagnosis.The detection of serum autoantibodies have been shown to correlate with disease activity and thus may be helpful in deciding treatment options for the patients.

Objective To investigated the association of serum level of growth arrest-specific protein 6 (Gas6) with the disease activity in patients with systemic lupus erythematosus. Methods The expression levels of Gas6 were detected by enzyme linked immunosorbent assay in 102 SLE patients and 67 healthy persons; and statistical methods were used to analyze the expressions of Gas6 in the patients with different clinical symptoms. Results The expression of Gas6 was significantly higher in the SLE group [54.59 (2.41 ~ 614.93) ng/mL] than in the healthy control group [19.22 (15.06 ~ 384.93) ng/mL, P < 0.05]. And serum Gas6 levels were significantly higher in the patients with dsDNA (+) , renal disorder , rash and vasculitis than the patients without symptoms (P < 0.05). The expression of Gas6 was correlated with SLEDAI significantly (r = 0.569, P < 0.01). Conclusions Gas6 plays an important role in the pathogenesis of SLE and it is an effective biomarker for the disease activity in patients with systemic lupus erythematosus.