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.

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

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 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

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 study the efficiency of surgical treatment on coarctation of the aorta and associated with heart defect.

METHODSFrom 1994 to 2001, 45 patients with aortic coarctation and associated with heart defect underwent surgical repair. They were divided into two groups: single-stage repair group (26 cases) and two-stage repair group (19 cases). There was mild or severe pulmonary hypertension in 23 cases (with mean pulmonary artery pressure being 56 mm Hg). There were two incisions used in first-stage group (single midline incision in 21 cases and left-side combined midline incision in 5 cases). The mean course for the second operation was 105 days in second-stage group.

RESULTSTwo patients died in each group. Twenty-four patients had not blood pressure difference between arm and leg after operation. The mean systolic blood pressure difference was less than 10 mmHg in 10 patients. Mean period of follow-up was 28.6 months. No patients died and had re-coarctation.

CONCLUSIONThe operative results showed no difference between single-stage and two-stage repair in surgical correction of aortic coarctation associated intracardiac defect. The left-side combined midline incision in single-stage operation was an effective and safe technique.

Adolescent ; Adult ; Aortic Coarctation ; surgery ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Heart Defects, Congenital ; surgery ; Humans ; Infant ; Male ; Treatment Outcome

OBJECTIVETo study the changes of intracellular interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) expressions in children with acute lymphoblastic leukemia (ALL) at different stages, and to examine the correlation between IL-6 and IFN-gamma in ALL children.

METHODSThe levels of intracellular IL-6 and IFN-gamma in venous blood lymphocytes were detected by flow cytometry in 42 children with ALL at diagnosis and at remission stage. Twenty healthy children were used as the controls.

RESULTSThe intracellular IL-6 level in ALL children at diagnosis was 81.74+/-9.31, which was much higher than that in the Control group (5.67 +/- 0.96 ) (P < 0.01). The intracellular IFN-gamma level in ALL children (1.31 +/- 0.32) was significantly lower than that in the Control group (1.46 +/- 0.49) (P < 0.01). However, the intracellular IL-6 level (27.52 +/- 3.40) decreased remarkably in ALL patients at remission stage (P < 0.01), but was still higher than that in the Control group (P < 0.01). In contrast, the intracellular IFN-gamma level (1.97 +/- 0.72) increased noticeably in ALL patients at remission stage, which was higher than that at diagnosis and the Control group (P < 0.01). A negative correlation was found between the intracellular IL-6 and the IFN-gamma levels in ALL patients (r=-0.476, P < 0.05).

CONCLUSIONSIntracellular IL-6 and IFN-gamma levels may be used as the markers for monitoring the response to treatment in ALL patients. There is a negative correlation between intracellular IL-6 and IFN-gamma levels in ALL children.

Adolescent ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; blood ; Interleukin-6 ; blood ; Leukocytes, Mononuclear ; chemistry ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology

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

Traditional Chinese medicinal resource survey method is time-consuming, strenuous, and having great human influence, the precision is not high enough. This paper, by using spatial information technology, carries on spatial sampling survey for wild medicinal plants resource for generous species to arrange the quadrat scientifically and estimate the suitable area, reserve precisely of medicinal plants. It not only improves the survey precision, but reduces the workload of field survey and provides scientific basis for survey method of pilot work on the fourth national traditional Chinese medicinal resource census.
Biodiversity ; Conservation of Natural Resources ; Ecosystem ; Humans ; Plants, Medicinal ; Spatial Analysis