1.Purification of Recombinant Fusion Protein Staphylokinase-Hirudin Expressed by Escherichia coli and Analysis of its Dimer
Gen-Shen ZHONG ; Ai-Ping YU ; Ji-De JIN ; Zhong-Hua JIANG ; Zu-Ze WU ;
China Biotechnology 2006;0(02):-
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.
2.Expression of Gastrin,Somatostatin,Proliferating Cell Nuclear Antigen and Fas-Ligand in Mucosa of Gastric Antrum in Children with Chronic Gastritis
xiao-zhi, XIE ; zhong-de, ZHANG ; zheng-jun, XI ; wen-wei, YU
Journal of Applied Clinical Pediatrics 2004;0(07):-
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.
3.Relationship between periodontal disease and rheumatoid arthritis.
Dai-zun ZHANG ; De-yu ZHONG ; Jing DENG ; Ji-bo WANG
West China Journal of Stomatology 2005;23(6):498-501
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
5.Effect of transient forebrain ischemia-reperfusion on the binding of brain-derived neurotrophic factor promoters to histone deacetylase 3 in hippocampus of rat and its mechanism
ZHANG Qian ; YAN De-ping ; SHI Jin-chao ; ZHONG Jin ; ZHOU Yang ; ZHAO Xin ; ZHANG Yu ; LI Jian-guo
Chinese Journal of Biologicals 2023;36(2):133-137
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.
6.Chinese Medicine Formula "Shenqi San" Extract Inhibits Proliferation of Human Lung Adenocarcinoma A549 Cells via Inducing Apoptosis
XIA YU ; SHI LU ; AI ZHONG-ZHU ; ZHANG DE-ZHONG ; LIU YAN-WEN ; YOU PENG-TAO
Journal of Huazhong University of Science and Technology (Medical Sciences) 2017;37(5):766-771
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.
7.Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase II beta subunit.
Zhai-yu PENG ; Xin-bing YU ; Zhong-dao WU ; Jin XU ; De WU ; Zi LI
Chinese Medical Journal 2004;117(9):1321-1325
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
8.Effects of the PI3K inhibitor,ZSTK474,on the proliferation and cell cycle of human melanoma A375 cells
Lei ZHANG ; Yu-Ling QIU ; Ying YING ; Yu-Xu ZHONG ; De-Xin KONG
Journal of International Pharmaceutical Research 2017;44(12):1118-1124
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.
9.Effects of 17β-estradiol and estrogen receptor modulator on the expression of cadherin and catenin in the uterus of ovariectomized rats.
Zhong-Rui DU ; Hong-Fang LI ; Wei ZHANG ; Zhi-Feng TIAN ; Xiao-Qing QIU ; Yan-Yu LU ; Qian-Kun LIANG ; Long-De WANG
Chinese Journal of Applied Physiology 2014;30(5):459-I
Animals
;
Cadherins
;
metabolism
;
Catenins
;
metabolism
;
Estradiol
;
pharmacology
;
Estrogen Receptor Modulators
;
pharmacology
;
Female
;
Ovariectomy
;
Rats
;
Uterus
;
drug effects
;
metabolism
10.Inhibition of glycogen synthase kinase 3b activity regulates Toll-like receptor 4-mediated liver inflammation.
Feng REN ; Hai-yan ZHANG ; Zheng-fu PIAO ; Su-jun ZHENG ; Yu CHEN ; De-xi CHEN ; Zhong-ping DUAN
Chinese Journal of Hepatology 2012;20(9):693-697
OBJECTIVETo determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4).
METHODSC57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively.
RESULTSThe phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 (control: 0.11 ± 0.05, inflammation: 0.85 ± 0.11, intervention: 0.43 ± 0.10; F = 2.67, P = 0.038), TNF-a, (control: 0.052 ± 0.012, inflammation: 8.11 ± 0.98, intervention: 3.9 ± 0.82; F = 4.13, P = 0.016), IL-6 (control: 0.22 ± 0.08, inflammation: 6.37 ± 0.81, intervention: 2.11 ± 0.63; F = 3.21, P = 0.024), and IL-1b (control: 0.12 ± 0.07, inflammation: 2.51 ± 0.62, and intervention: 1.28 ± 0.33; F = 2.22, P = 0.030).
CONCLUSIONInhibition of GSK3b selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells (liver macrophages). This process may be one of the mechanisms underlying the ability of GSK3b to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.
Animals ; Cells, Cultured ; Cytokines ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Inflammation ; metabolism ; pathology ; Lipopolysaccharides ; adverse effects ; Liver ; pathology ; Macrophages ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; Toll-Like Receptor 4 ; metabolism