The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.

Objective To investigate the expression of immunohistochemistry of gastrin(GAS),somatostatin(SS),proliferating cell nuclear antigen(PCNA) and Fas-ligand(Fas-L) in the sinus ventriculi of children with pediatric gastritis and to explore the significance of their expression in the pathogenesis of pediatric chronic gastritis.Methods Fifty cases of the sinus ventriculi mucosa samples were enrolled in 3 groups:chronic gastritis,helicobacter pylori(Hp) positive(group A,n=20);chronic gastritis,Hp negative(group B,n=19);control group,normal sinus ventriculi mucosa,Hp negative(group C,n=11).Immunohistochemistry En Vision were carried out including GAS,SS,PCNA and Fas-L.Results In the expression of GAS and SS,the values of group A and B were comparatively higher than those of group C,but there was no significant difference among them in statistics.In the expression of PCNA,the value of group A was comparatively higher and that of group B.The value difference between 2 groups was significant(P=0.019);in the expression of Fas-L,no significant difference was found among these 3 groups.Conclusions Expressions of GAS and SS both increase in children with chronic gastritis and maybe the increase of GAS and SS play a role in the pathogenesis of pediatric chronic gastritis;Hp infection promotes the multiplication of the sinus ventriculi membrana mucosa epithelium cell in pediatric chronic gastritis.

OBJECTIVETo study a population of rheumatoid arthritis patients and determine the extent of periodontal disease in these patients, in order to investigate the relationship between periodontal disease and rheumatoid arthritis.

METHODSThe experimental group was composed of 70 patients with rheumatoid arthritis and the control group consisted of 70 age- and gender-matched individuals without rheumatoid arthritis. The relationship between periodontal status in rheumatoid arthritis and control groups as well as the relationship between periodontal status and rheumatological findings in patients were analyzed.

RESULTSThe percentage of periodontal disease was statistically significant between experimental and control group (P < 0.01). The difference of average number of missing teeth and bleeding on probing in the experimental group and control group were not statistically significant (P >0.05). There were more number of periodontal disease index 5 or 6 in experimental group than in control group ( P < 0.05). Rheumatoid arthritis patients with moderate to severe bone loss had deeper degree of morning stiffness, erythrocyte sedimentation rate levels and serum C-reactive protein levels than patients with no or mild bone loss.

CONCLUSIONIndividuals with rheumatoid arthritis are more likely to experience periodontal disease compares to healthy subjects. They are also very likely to suffer from moderate to severe periodontitis.

Adult ; Arthritis, Rheumatoid ; Blood Sedimentation ; C-Reactive Protein ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Periodontal Diseases ; Periodontitis

Aged ; Aged, 80 and over ; Atherectomy, Coronary ; instrumentation ; methods ; Female ; Humans ; Male ; Treatment Outcome

Transient forebrain ischemia-reperfusion(I/R);Brain-derived neurotrophic factor(BDNF);Histone deacetylase 3(HDAC3);Hippocampus Objective To evaluate the effect of transient forebrain ischemia-reperfusion(I/R)on the binding of brainderived neurotrophic factor(BDNF)promoters to histone deacetylase 3(HDAC3)in the hippocampus of rat and investigate its mechanism.Methods The I/R model of SD rats(I/R group)was established by Pulsinelli four-vessel clamping method,and sham operation group(Sham group)was set at the same time,which were observed for the survival of neurons in the hippocampus of rats by Nissl staining,detected for the binding of BDNF promoters(Bdnf-p1,Bdnf-p2,Bdnf-p4 and Bdnf-p6)to HDAC3 by chromatin immunoprecipitation(ChIP)and determined for the expression of brain derived neurotrophic factor antisense(BDNF-AS)by qPCR.Results Compared with Sham group,the quantity of neurons in hippocampal CA1 region of rats decreased significantly in I/R group,while those in CA3 region and DG region showed no significant changes.The binding levels of Bdnf-p1 and Bdnf-p2 to HDAC3 in hippocampal CA1 region decreased significantly in I/R Group(t = 2.575 and 2.241 respectively,each P<0.05),while there was no significant difference in the binding levels of Bdnf-p4 and Bdnf-p6 to HDAC3(t = 1.033 and 0.348 respectively,each P>0.05);The binding levels of Bdnf-p1 and Bdnf-p2 to HDAC3 in CA3 region increased significantly(t = 12.600 and 3.191,P<0.001 and<0.05,respectively),while the binding level of Bdnf-p6 to HDAC3 decreased significantly(t = 4.029,P<0.05)and no significant difference was observed in the binding level of Bdnf-p4 to HDAC3(t = 0.175,P>0.05);In DG region,the binding level of each BDNF promoter to HDAC3 showed no significantly difference(t = 0.630 ～ 1.687,each P>0.05).Meanwhile,the expression level of BDNF-AS in hippocampal CA1 region of rats decreased significantly(t = 2.560,P<0.05),but increased significantly in hippocampal CA3 and DG regions(t = 3.543 and 3.637 respectively,each P<0.01)in I/R group.Conclusion I/R showed a significant effect on the binding level of BDNF promoter to HDAC3 in rat hippocampus,which may play a role by changing the expression level of BDNF-AS.

The main purpose of this study was to investigate the active components of the Chinese medicine formula Shenqi San (SS) by high performance liquid chromatography with diode array detector and electrospray ionization-hybrid quadrupole time-of-flight mass spectrum (HPLC-DAD-ESI-QTOF-MS),and demonstrate the anticancer mechanism of SS on human lung adenocarcinoma A549 cells by evaluating the cell proliferation and apoptosis induction.The chloroform extraction of SS (CE-SS) was extracted from SS,while HPLC-DAD-ESI-QTOF-MS assay was performed to identify components of CE-SS.MTT assay was used to quantify the proliferation of A549 cells with the treatment of CE-SS.Apoptosis analysis was carried out by detecting phosphatidylserine (PS) externalization using the Annexin V-FITC Apoptosis Detection Kit and the stained cells were analyzed with a flow cytometer.DAPI staining assay was carried out to observe morphological characteristics of apoptotic cells.Western blotting was used to detect the expression of important signaling proteins including caspase-3,-8,-9,p53,Bax and Bcl-2.Eight compounds were identified through HPLC-DAD-ESI-QTOF-MS analysis and 3-pyridine carboxylic acid,barbatin C,scutebarbatine F and barbatine D might be the main compounds responsible for the antitumor effect of CE-SS.CE-SS suppressed the proliferation of lung cancer A549 cells in a time-and dose-dependent manner.By Annexin V-FITC/PI double staining,we found that treatment with CE-SS induced apoptosis in A549 cells.After 24-h exposure to CE-SS,the expression of cleaved-caspase-9,cleaved-caspase-8 and cleaved-caspase-3 protein was activated,the expression of p53 protein increased while the ratio of Bax/Bcl-2 also increased.This study identified the eight compounds of CE-SS,and demonstrated their anticancer effect on human lung adenocarcinoma A549 cells via induction of apoptosis.

BACKGROUNDNowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase II beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli).

METHODSThe ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot.

RESULTSA 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase II beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E. coli JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum.

CONCLUSIONThe full-length cDNA sequences encoding S. japonicum casein kinase II beta subunit were firstly sequenced, cloned, and expressed in E. coli.

Amino Acid Sequence ; Animals ; Base Sequence ; Blotting, Western ; Casein Kinase II ; chemistry ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; isolation & purification ; Escherichia coli ; genetics ; Molecular Sequence Data ; Rabbits ; Schistosoma japonicum ; enzymology ; genetics

Objective To investigate the effect and molecular mechanisms of phosphatidylinositol-3 kinase(PI3K)inhibitor ZSTK474 on human melanoma A375 cells in vitro. Methods The effect of ZSTK474 on the proliferation of A375 cells was deter?mined by MTT assay.Flow cytometric analysis was carried out to examine effect of ZSTK474 on the cell cycle of A375 cells.Western-blot was conducted to evaluate the effect of ZSTK474 on the expression of the cell cycle related proteins,cyclin B1 and cdc2.Chou-Talalay method was used to evaluate the combination of ZSTK474 with PD0332991.Results In the MTT assay,ZSTK474 inhibited the proliferation of A375 cells in a dose-dependent manner with the IC50value of 1.535 μmol/L.Furthermore,ZSTK474 arrested the cell cycle progression of the A375 cells at the G2/M phase via downregulating the expression of cyclin B1 and cdc2 at 1 and 5 μmol/L. In the synergistic assay,the combination of ZSTK474 with PD0332991 in the ratio 8×IC50 ZSTK474:1×IC50 PD0332991showed a synergistic ef?fect,with the combination index(CI)values of 0.463 ± 0.113,0.658 ± 0.009 and 0.941 ± 0.034 for ED50、ED75and ED90,respectively. Conclusion ZSTK474 could inhibit the proliferation of A375 cells and arrest the cell cycle at the G2/M phase.The combination of ZSTK474 with PD0332991 could exert a synergistic effect.The precent result has revealed that the PI3K inhibitor ZSTK474 is likely to be applied alone or in combination with the CDK4/6 inhibitor PD0332991 for the human melanoma therapy.

OBJECTIVEDamage control surgery (DCS) deals with the complex surgical problems by stages. This study investigated the application of DCS in serious pediatric abdominal surgery.

METHODSThe clinical data of 49 children with serious abdominal diseases (age: 4 months to 10 years) were retrospectively studied. Of them, 32 children underwent damage control surgery (DCS) and 17 children underwent conventional operation. The preoperative critical severity score (CSS), postoperative temperature, blood pH and prothrombin time (PT), and the treatment outcome were compared between the DCS and the conventional operation groups.

RESULTSNo significant difference was found in the preoperative CSS between the two groups. There were significant differences in postoperative blood pH and PT values between the two groups (p<0.05). As for postoperative temperature, there was no statistical difference between the two groups, yet the tendency of temperature recovery in the DCS group was milder than that in the conventional operation group. Twenty-seven children (84.4%) were successfully cured in the DCS group, while 9 children (52.9%) in the conventional operation group (p<0.05).

CONCLUSIONSThe curative effect of DCS surpasses the conventional operation in children with serious abdominal diseases, suggesting that DCS is of value in the management of serious pediatric abdominal diseases.

Abdomen ; surgery ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Prothrombin Time ; Retrospective Studies ; Surgical Procedures, Operative ; methods

In order to establish HPLC fingerprint of Liuwei Dihuang soft capsule, and to provide certain reference for an quality control of it, the HPLC method was performed on an Agilent C18 (4.6 mm x 250 mm, 5 μm) column with acetonitrile-0.02% trifluoroacetic acid as mobile phase, gradient elution volume flow of 1.0 mL x min(-1), column temperature was 30 degrees C, detection wavelength: 0-60 min, 238 nm, 60-70 min, 210 nm. The software for chromatographic fingerprint was applied to analysis different batches of Liuwei Dihuang soft capsule samples. Sixteen mutual peaks were selected as the fingerprint peaks in 12 samples with loganin as the reference peak, and all of the detected peaks were separated effectively. Cluster analysis (HCA) and similarity analysis (SA) were done based on data of 12 samples clustering analysis of 12 batches of samples were divided into 2 categories. Including 7 for the first class, the rest was second, similarities calculated by SA were all above 0.92, indicating a good similarity between the reference and twelve batches of samples, also, the analysis results of HCA and SA basically the same. This method is simple with good precision, repeatability and stability, and provides the basis for Liuwei Dihuang soft capsule quality control.
Capsules ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Quality Control