Objective To investigate cholecystokinin (CCK),carbachol, and vasoactive intestinal peptide(VIP)stimulating peptide chain elongation in mouse pancreatic acini in vitro and its molecular mechanism. Methods ~3H-lecucine incorporation assay was used to measure the basal and secretagogues-stimulated pancreatic acini elongation rates. Western blot was applied to analyse the effect of phosphorylation of the elongation factor 2 (eEF2) and the eEF2 kinase. MEK inhibitor (PD98059), SAPK/p38 inhibitor (SB202190), and mTOR inhibitor (rapamycin) were used to respectively block MEK, SAPK/p38, and mTOR intracellular pathways or the phosphatase inhibitor (calyculin A) pretreatment before CCK treatment. Results All secretagogues except VIP increased the peptide chain elongation in mouse pancreatic acini in vitro. All secretagogues except VIP inhibited the phosphorylation level of eEF2 on Thr-56 and increased the phosphorylation level of eEF2K on Ser-366, which might correlate with their activation status. MEK inhibitor PD98059 partially reversed the dephosphorylation of eEF2 induced by CCK, as did treatment p38 MAPK inhibitor SB202190, mTOR inhibitor rapamycin, and the phosphatase inhibitor calyculin A.Conclusion CCK increases peptide chain elongation via inducement of dephosphorylation of eEF2 and eEF2 kinase phosphorylation in pancreatic acini in vitro. CCK-induced dephosphorylation of eEF2 in pancreatic acinar cells involves MEK, SAPK/p38, and mTOR, the three intracellular pathways.

The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.

Objective In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function.Methods Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. Results SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239~(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1~(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1~(-/-) males than wild-type littermates at 15 weeks of age. SH2B1~(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5~(th) week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates.Conclusion SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.

Objective To identify lung adenocarcinoma-associated antigens by using Serologic Proteome Analysis(SERPA),an approach which combined conventional proteome analysis with serological screening.Methods SERPA of four human lung adenocarcinoma tissues were performed.The Western blot imaging films which reacted with autologous patient serum and with control normal serum were obtained and the differential reacting protein spots were recognized.Results Well-resolved,reproducible 2-DE Western blot imaging films of human lung adenocarcinoma reacted with autologous patient sera and the control sera were obtained.Totally(27?5) differentially expressed proteins which only were reactive with lung adenocarcinoma patient sera were found.Some differentially expressed proteins were identified by peptide mass fingerprint(PMF).Some of the proteins were the products of oncogenes,and others were involved in the regulation of cell cycle and signal transduction.Conclusion These results will provide scientific foundation on screening the molecular biomarker for the diagnosis,treatment,and prognosis of the lung adenocarcinoma.

Objective To observe the expression and influence of SH2-B in hepatocarcinoma,and to investigate the molecular mechanisms of canceration in hepatocarcinoma.Methods By using SABC imunohis-tochemistry,the expressions of SH2-B were detected in 27 cases of hepatitis,29 cases of hepatocirrhosis and 47 cases of hepatocarcinoma.Hepatocarcinoma cell (HepG)2 with a low-expressed SH2-B was selected using immunofluorescence assay.There were 3 groups:the transfected group (transfected with pcDNA3.1 -SH2-B), the vector group (transfected with pcDNA3.1 )and the blank group (without transfection).After gene transfec-tion,SH2-B expression was detected by Western blotting;cell proliferation was measured by MTT assay;cell colony was counted by colony formation test;and cell cycle was analyzed by flowcy tometer.Results The posi-tive rate of SH2-B in hepatocarcinoma (95.7%)was significantly higher than 55.2% in hepatocirrhosis (χ2 =1 8.64,P <0.01 )and 25.9% in hepatitis (χ2 =40.01 ,P <0.01 ).After being transfected with pcDNA 3.1 -SH2-B,SH2-B expression dramatically increased in HepG2 cells.After cultured for 48 h,the average optical density value of the transfected group was 1 .1 2 ±0.1 9,obviously higher than 0.45 ±0.1 1 in the vector group (t =-31 .55,P <0.01 ),which indicated that cells proliferation was significantly enhanced after being trans-fected with SH2-B.The cell colony numbers of the transfected group was 1 66 ±1 4,significantly higher than
82 ±8 in the vector group (t =-20.33,P <0.01 )and 78 ±9 in the blank group (t =-1 9.64,P <0.01 ), which indicated that the cell colony numbers increased after being transfected with SH2-B.The S stage cells of the transfected group was (45.7 ±5.8)%,significantly higher than (1 9.4 ±4.7)% in the vector group (t =-20.33,P <0.01 )and (20.5 ±5.1 )% in the blank group (t =-34.69,P <0.01 ),which indicated that SH2-B could enhance promote cell cycle of HepG2 cells.Conclusion The expression of SH2-B in hepatocar-cinoma is high,and it may be involved in the canceration of hepatocarcinoma though promoting cell cycle,cell proliferation and cell transformation.

To screen for serum biomarkers for lung squamous carcinoma, two-dimensional gel electrophoresis (2-DE) was performed to separate serum proteins from healthy individuals and stage 1 lung squamous carcinoma(LSC) patients, respectively. PDquest software was used to analyze 2-DE images, and the differential serum protein spots between the healthy individuals and LSC patients were identified by ESI-Q-TOF MS/MS. Then Western blot and immunohistochemistry were used to detect the expression levels of haptoglobin-2(HP-2), one of the differential proteins, in the sera and tumor tissues in the patients with LSC, respectively. 2-DE maps of serum proteins from healthy individuals and stage 1 LSC patients were established. Ten differential serum protein spots were detected, four proteins of which were identified by MS/MS. Western blot showed that the serum level of HP-2 in the LSC patients was significantly higher than that in healthy individuals, but was not associated with LSC staging. Immunohistochemistry showed that the expression level of HP-2 in the LSC tissues was significantly higher than that in the normal bronchial epithelial tissues adjacent to tumors. The results indicated that serum HP-2 protein is a candidate biomarker for LSC, and might be useful for diagnosis of LSC. Up-regulation of HP-2 in the LSC tissues may contribute to the high serum level of HP-2 in the patients.

OBJECTIVE: To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels. METHODS: The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot. RESULTS: Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression. CONCLUSION SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Hypothalamus ; metabolism ; Insulin ; blood ; Insulin Resistance ; Leptin ; blood ; Mice ; Mice, Inbred C57BL ; Neuropeptide Y ; metabolism ; Obesity ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; metabolism ; RNA, Messenger ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; metabolism

Aim To investigate the role of cholesterol in the regulation of lipid raft and the function of platelets.Methods Using in vitro incubation of methyl β-cyclodextrin(MβCD)and in vivo elevation of peripheral blood total cho-lesterol levels to remove and load platelet cholesterol,respectively.Cholera toxin B staining combined with flow cytometry was used to detect platelet lipid raft content,fluorescence antibody staining combined with flow cytometry was used to detect the expression levels of P-selectin and activated integrin α Ⅱ bβ3,annexin Ⅴ labeling combined with flow cytometry was used to detect the level of phospholipid efflux,in vitro experimental system and rat tail bleeding experiment were used to detect platelet aggregation ability.Results The content of lipid raft on B lymphocytes decreased with the removal of cholesterol,while in vitro incubation of MβCD to remove platelet cholesterol significantly increased its lipid raft level(P＜0.05).Consistent with this,in vivo cholesterol loading increased the lipid raft content of B lymphocytes but decreased the lipid raft content of platelets(P＜0.05).The increase in lipid raft after removing cholesterol was not conducive to platelet activation and aggregation function.In vivo cholesterol loading downregulated platelet lipid raft content(P＜0.05),enhanced its ability to respond to low concentration stimulant for activation aggregation and coagulation,and this enhancing effect disappeared after cholesterol removal.Conclusion Platelet cholesterol is a key regulator of platelet lipid raft content and platelet function,which can negatively regulate lipid raft,promote platelet activation,and enhance their coagulation function.