Method of isolation and purification of recombinant hepcidin was described, and the bioactivity of the protein was assayed in this paper.The oxidation of his-hepcidin was carried out in the cysteine-cystine system, and the multimers were removed through gel filtration under denaturation condition.Then the protein was refolded by continuous dilution and digested by enterokinase.The total yield of his-hepcidin before enterokinase cleavage is 50%, and the purity is above 95%.Through agar diffusion assay, the recombinant hepcidin displayed obvious antibacterial activity against B.subtilis.The LC-ESI-MS analysis of recombinant hepcidin showed that the measured molecular weight accorded with the calculated molecular weight, and the CD spectrum indicated that the secondary structure of recombinant hepcidin is similar with native hepcidin.

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

To study the mechanism of thrombogenesis and search new anti-thrombotic agent, the cDNA for human vWF A1 domain was high-level expressed in E. coli and recombinant protein of vWF A1 with biologic activity was obtained. The gene encoding A1 domain was amplified by PCR from plasmid containing full length cDNA of human vWF. After confirming by DNA sequencing analysis, the recombinant expression plasmid pQE31-vWF/A1 was constructed and introduced into E. coli M15 strain, then induced by IPTG; the expressed protein was purified with Ni-NTA agarose, identified by Western blotting. The results showed that the 854 bp DNA fragment was obtained by PCR from the plasmid containing full length cDNA for human vWF and its sequence was identical to the published sequence. High level expression of A1 protein was yielded after 5 hour-induction, which amounted to 30% of total bacteria protein in inclusion body. Western blot demonstrated it possessed good antigenicity and high specificity. It is concluded that cDNA for vWF/A1 had been cloned successfully, high level expression of A1 protein was achieved in E. oli. This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Escherichia coli ; genetics ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; analysis ; von Willebrand Factor ; analysis ; biosynthesis ; genetics

To characterize the background current in fetal human nasopharyngeal epithelial cells and clarify its relationship with volume activated Cl(-) currents (I(Cl,vol)), whole-cell patch clamp and cell imaging techniques were employed. Under isotonic conditions, a background current [(5.9+/-2.1) pA/pF at +80 mV, n=21] was detected. The current presented a weak outward rectification and a negligible time-dependent inactivation. The current-voltage relationship showed that the reversal potential of the background current [(-0.73+/-1.7) mV, n=21] was close to the calculated equilibrium potential for Cl(-)(-0.9 mV). Application of extracellular hypertonic stimulation (440 mOsmol/L) suppressed the current by (59.6+/-7.1)% and the inhibition was reversible after returned to isotonic conditions. Bathing the cells in hypotonic solution (160 mOsmol/L) induced a volume-sensitive Cl(-) current. The Cl(-) channel blockers, tamoxifen (20 micromol/L) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (100 micromol/L), inhibited the background current by (74.0+/-5.2)% (P<0.01, n=5) and (60.9+/-8.9)% (P<0.01, n=6) at +80 mV and increased basal cell volume by (107.7+/-2.9)% (P<0.01, n=25) and (104.4+/-2.4)% (P<0.01, n=19), respectively. The data indicate that Cl(-) current is an important component of the background current in fetal human nasopharyngeal epithelial cells. The background Cl(-) current is involved in volume activated Cl(-) current and basal cell volume regulation.
Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Electrophysiology ; Epithelial Cells ; cytology ; metabolism ; physiology ; Fetus ; Humans ; Nasopharynx ; cytology ; Nitrobenzoates ; pharmacology ; Patch-Clamp Techniques ; Tamoxifen ; pharmacology

OBJECTIVETo explore the incidence and angiographic features of exercise-induced ST-segment elevation in patients without prior myocardial infarction.

METHODSExercise-induced ST-segment elevation occurred in 15 out of 4601 consecutive patients without prior myocardial infarction underwent treadmill exercise testing during a 2-year period. The coronary angiographic features of the 15 patients (13 males, aged between 40 - 75 years) were analyzed.

RESULTSCoronary angiography revealed one hemodynamically relevant stenotic vessel in 6 patients, two hemodynamically relevant stenotic vessels in 6 patients, three hemodynamically relevant stenotic vessels in 3 patients. Left anterior descending (LAD) coronary artery was affected in 12 patients. Left main coronary artery (LMCA) stenosis was evidenced in 1 patient and right coronary artery stenosis in 7 patients. Severe (90% - 100%) occlusions were visualized in 8 out of 13 patients with LAD or LMCA lesions. Elevated ST-segment leads were consistent with the ischemic area where the blood supply of myocardium was affected by diseased vessels.

CONCLUSIONSThe incidence of exercise induced ST-segment elevation in patients without prior myocardial infarction is very low and mostly due to severe fixed coronary artery stenosis, especially in LAD. The location of ischemic myocardium can be suggested by ST-segment elevation leads during exercise.

Adult ; Aged ; Coronary Angiography ; Coronary Artery Disease ; diagnosis ; physiopathology ; Electrocardiography ; Exercise Test ; Female ; Humans ; Male ; Middle Aged

OBJECTIVESTo analyze the prognostic factors for elderly patients with pancreaticoduodenectomy for periampullary tumor.

METHODSA retrospective analysis of the prognostic factors for the mortality rate was made in 127 elderly patients within 30 days of pancreaticoduodenectomy for periampullary tumor from January 1985 to November 2006 Chi-squared test, Fisher's exact test, t-test were used.

RESULTSThe prognostic factors for the first-month mortality rate in elderly patients with pancreaticoduodenectomy included time length of the operation, operative hemorrhage, postoperative hemorrhage, pulmonary infection, and postoperative TP.

CONCLUSIONSAn overall consideration should be paid to the factors that affect the prognosis of elderly patients with pancreaticoduodenectomy for periampullary tumor during the perioperative period. The security of the patients can be promoted by controlling these prognostic factors.

Aged ; Ampulla of Vater ; Common Bile Duct Neoplasms ; surgery ; Female ; Humans ; Male ; Middle Aged ; Pancreaticoduodenectomy ; mortality ; Prognosis ; Retrospective Studies ; Survival Analysis

OBJECTIVETo study the inhibitory effects of Panax Notoginseng Saponins(PNS) on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons.

METHODApoptosis were measured by flow cytometry, intracellular free calcium concentration([Ca2+]i) was measured with confocal laser scanning microscopy, morphological changes and neuronal necrosis were observed with fluorescence microscope, and meanwhile the leakage of lactic dehydrogenase(LDH) was measured.

RESULTHypoxia/hypoglycemia cultures for 5 hours and reoxygenation induced neuronal apoptosis and necrosis, and significantly increased neuronal [Ca2+]i and the leakage of LDH. The effects were increased with the extending time of reoxygenation. PNS has could significantly decrease the percentage of neuronal apoptosis and necrosis, and reduce neuronal [Ca2+]i and the leakage of LDH.

CONCLUSIONPNS has inhibitory effect on neuronal apoptosis. This effect might be related to its effect of decreasing intracellular free calcium concentration.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Fetus ; Ginsenosides ; isolation & purification ; pharmacology ; Hippocampus ; cytology ; Hypoglycemia ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Neurons ; cytology ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar

OBJECTIVETo investigate the association of peripheral and central blood pressure with the alpha-adducin Gly460Trp polymorphism in Chinese.

METHODSWe randomly selected 6 villages from JingNing County, ZheJiang Province. We invited nuclear families to take part in our study. We measured each participant's blood pressure at the non-dominant arm by means of a standard mercury sphygmomanometer at subjects' homes. Five consecutive readings were averaged for analysis. Central blood pressures were obtained by use of SphigmoCor pulse wave analysis system. The observers administered a standardized questionnaire to collect information on smoking habits, alcohol consumption and use of antihypertensive drugs. Venous blood was sampled and the adducin genotype was determined by restrictive fragment length polymorphism (RFLP).

RESULTSFour hundred and forty-two subjects included 230 (52.0%) women, and 116 (26.2%) hypertensive patients, of whom 49 (11.1%) took antihypertensive drugs. The frequencies of alpha -adducin GlyGly, GlyTrp and TrpTrp genotypes were 21.3%, 54.5% and 24.2%, respectively. There was no association between the alpha-adducin Gly460Trp polymorphism and peripheral systolic and diastolic blood pressure and pulse pressure. However, both before and after adjustment for sex, age, age(2), body-mass index, current smoking, alcohol intake, and antihypertensive treatment, the alpha-adducin polymorphism was significantly (P < 0.02) associated with central systolic blood pressure and central pulse pressure. After adjustment, central systolic blood pressure (+/- SE) averaged 122.5 +/- 3.5, 114.1 +/- 1.5 and 109.1 +/- 1.8 mm Hg (P = 0.01) in the GlyGly, GlyTrp and TrpTrp subjects, respectively. The corresponding values for central pulse pressure were 39.4 +/- 1.3, 36.4 +/- 1.0 and 32.9 +/- 0.9 mm Hg (P = 0.002), respectively.

CONCLUSIONSIn the JingNing population, the adducin 460Trp allele was associated with lower levels of central systolic pressure and pulse pressure.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Blood Pressure ; Calmodulin-Binding Proteins ; genetics ; Child ; China ; epidemiology ; Female ; Gene Frequency ; Genotype ; Humans ; Hypertension ; epidemiology ; genetics ; physiopathology ; Male ; Middle Aged ; Pedigree ; Polymorphism, Single Nucleotide ; Young Adult

This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.
Blood Platelets ; metabolism ; Blotting, Western ; Escherichia coli ; genetics ; Humans ; Integrin alpha2beta1 ; Platelet Membrane Glycoproteins ; biosynthesis ; genetics ; Protein Binding ; Receptors, Collagen ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo investigate effects of GSTM1 and GSTT1 gene polymorphisms on serum lipid and apopoprotein levels in healthy normolipidemic and endogenous hypertriglyceridemic subjects.

METHODSTwo hundred and thirty-seven healthy normolipidemic and 102 endogenous hypertriglyceridemic subjects from a population of Chinese Han nationality in Chengdu area were studied using the multiplex polymerase chain reaction (PCR). Serum lipids were measured by enzymatic kits and apolipoproteins AⅠ, AⅡ, B100, CⅡ, CⅢ and E were measured by the RID kits.

RESULTSThe non-null and null genotype frequencies for GSTM1 site were 39.2% and 60.8% in the control group, respectively, and 47.6% and 52.4% in the HTG group, respectively. The non-null and null genotype frequencies for GSTT1 site were 51.5% and 48.5% in the control group, respectively, and 57.3% and 42.7% in the HTG group, respectively. The GSTM1 and GSTT1 genotype frequencies in HTG subjects were not different from those in the controls, respectively (P>0.05). However, in control group subjects with both null genotypes (GSTT1- and GSTM1-) showed the lowest plasma HDL-C levels (1.29±0.30 mmol/L), whereas those with each of the other three genotype combinations showed relatively higher HDL-C levels. There was significant difference of HDL-C levels between subjects with GSTT1-/GSTM1-and those with GSTT1+/GSTM1-(P<0.05). Similar result was not observed in HTG group. No significant changes of lipid and lipoprotein levels were observed in either GSTM1 or GSTT1 polymorphism alone in control or HTG group.

CONCLUSIONThe present study provides an evidence that the presence of double deletion genotypes is associated with low HDL-cholesterol levels in normal Chinese subjects. However, these polymorphisms are not associated with lipid levels in endogenous hypertriglyceridemia in Chinese population of Chengdu area.