OBJECTIVEE23K polymorphism in KCNJ11 gene is associated with cardiovascular disease and diabetes. In order to explore the mechanism of E23K correlation to related diseases, the effect of E23K polymorphism in KCNJ11 gene on membrane current was investigated.

METHODSThe exon of KCNJ11 was obtained by PCR amplification and the G-->A mutation was completed by overlap extension PCR. The sequences of KCNJ11 exon contained 23E or 23K was inserted into pcDNA3.1/CT-GFP vector respectively. The recombinant plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were transfected into HEK293T cells by lipofectamine and the membrane current density was determined by whole-cell patch clamp technique.

RESULTS1,173 bp sequences of KCNJ11 gene's exon were amplified by PCR and the recombinant expression plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were constructed successful. Positive and negative currents were detected in HEK293T cells transfected with difference plasmid by whole-cell patch clamp technique. Results showed that the reversed voltage was 50mV. The current in HEK293T cells with pcDNA3.1-KCNJ11(E) was significantly greater than that with pcDNA3.1-KCNJ11(K) (P < 0.05, n = 10).

CONCLUSIONThe polymorphism of E23K in exon of KCNJ11 gene changed the membrane currents in HEK293T cells. It could be an experiment support for the possible mechanism between the locus and related diseases.

Exons ; HEK293 Cells ; Humans ; Membrane Potentials ; Patch-Clamp Techniques ; Polymorphism, Genetic ; Potassium Channels, Inwardly Rectifying ; genetics

A highly phytase-producing strain B.licheniformis LL8 was obtained by several mutagenesis of UV with B.licheniformis as starting strain.The new strain produced about two folds of phytase activity as compared with the starting strain. The production performance of the strain was stable. The cultivation conditions were optimized by single factor and orthogonal experiment. When the mutant B.licheniformis LL8 was cultivated at 55℃, initial pH 7.5 with the inoculation size of 10% for 30h in WBE medUm, the phytase activity was up to 2268.4U/mL.

OBJECTIVETo explore the correlation of the gene polymorphisms of Toll-like receptor 2 ( TLR2) and TLR4 with the susceptibility and recurrence of condyloma acuminatum (CA).

METHODSUsing Snapshot, we detected the gene polymorphisms of TLR2 597(T/C), 1350(T/C), 15607(A/G), and 2258(G/A) and TLR4 896(A/G) and 1196(C/T) in the peripheral blood of 140 CA patients and 105 HPV-negative controls. We made comparisons between the CA patients and controls as well as between the cases of recurrent CA and those of non-recurrence at 6 months after treatment.

RESULTSThere were 72, 48, and 20 cases of genotype TT, TC, and CC of TLR2 597 (T/C), respectively, in the CA patients, as compared with 71, 31, and 3 cases in the controls. The gene frequency of mutant C was 31. 43% in the patients, significantly higher than 17.62% in the controls (χ2 = 12.04, P < 0.01), and it was 38.68% in the recurrent cases, remarkably higher than 27.01% in the non-recurrent cases (χ2 = 4.16, P < 0.05). There were 74, 49, and 17 cases of genotype TT, TC, and CC of TLR2 1350( T/C), respectively, in the CA patients, as compared with 73, 29, and 3 cases in the controls. The gene frequency of mutant C was 29. 64% in the patients, significantly higher than 16. 67% in the controls (χ2 =11.05, P < 0.01), and it was 36.79% in the recurrent cases, markedly higher than 25. 29% in the non-recurrent cases (χ2 = 4.18, P < 0.05). There were 44, 66, and 30 cases of genotype AA, AG, and GG of TLR2 15607(A/G), respectively, in the CA patients, as compared with 26, 58, and 21 cases in the controls. There was no significant difference in the gene frequencies of mutant G between the two groups (χ2 = 0.33, P > 0.05). No mutant genes of TLR2 2508 (G/A) or TLR4 896(A/G) and 1196(C/ T) were detected in either the CA patients or the controls. Linkage disequilibrium analysis showed a tight linkage between TLR2 597 (T/C) and 1350(T/C) (D' = 1, r2 = 0.93).

CONCLUSIONTLR2 597(T/C) is tightly linked to 1350(T/C), which is correlated with both the susceptibility and the recurrence of condyloma acuminatum.

Aged ; Case-Control Studies ; Condylomata Acuminata ; genetics ; Gene Frequency ; Genetic Linkage ; Genetic Predisposition to Disease ; Genotype ; Humans ; Polymorphism, Genetic ; Recurrence ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics

OBJECTIVETo observe the intervention of Shenkangling Decoction (SD) on the renal injury of primary nephrotic syndrome (PNS) children patients of Shen deficiency blood stasis syndrome (SDBSS) and to explore its mechanism.

METHODSTotally 65 PNS children patients were randomly assigned to the combined group (33 cases, treated by SD +Western medicine) and the Western medicine group (32 cases, treated by Western medicine). Meanwhile, 30 healthy children were recruited as the healthy control group from the medical examination center. Those in the Western medicine group were treated with prednisone (5 mg per tablet) at the daily dose of 1.5 -2.0 mg/kg till two weeks after their urine protein turned to negative. Then the dosage was reduced once daily per every other day. The therapeutic course lasted for more than 1 year. For those with no effect of prednisone or partial effect, cyclophosphamide intravenous pulse therapy was additionally applied for 2 successive days per week, a total of 6 times, or they took cyclosporine A. Patients in the combined group additionally took SD while starting treatment of prednisone. SD was decocted in water for oral dose, once daily, taken in two portions until 2 months after prednisone was discontinued. Efficacy was evaluated based on serum levels of chemotactic factor CXCL16, disintegrin metalloproteinase 10 ( ADAM10 ), disintegrin metalloproteinase 17 (ADAM17), albumin (ALB), total cholesterol (TC), and 24-h urine protein excretion (UPE) detected by ELISA before and after treatment.

RESULTSCompared with before treatment in the same group, levels of CXCL16, ADAM10, ADAM17, TC, and 24-h UPE were significantly lower in the two treatment groups (P <0. 01). Compared with the control group, levels of CXCL16, ADAM10, ADAM17, TC, and 24-h UPE significantly increased, and the serum ALB level decreased in the two treatment groups (P <0.01). Compared with the Western medicine group at the same time point, levels of CXCL16, ADAM10, ADAM17, TC, and 24-h UPE significantly decreased in the combined group. The 1 -year recurrence rate and the recurrence times decreased in the combined group (P <0.01). The complete remission rate increased in the combined group (P <0.01).

CONCLUSIONSD could effectively improve the clinical prognosis of PNS children patients possibly by reducing the release of inflammatory mediators such as CXCL16, ADAM10, and ADAM17, decreasing UPE and the TC level, and elevating the serum ALB level.

Child ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; Nephrotic Syndrome ; drug therapy ; Prednisone ; Syndrome

There have been very few studies on the effect of Gualou Xiebai Banxia decoction combined with Xuefu Zhuyu decoction in inhibiting apoptosis in myocardial ischemial injury caused by coronary heart disease. In this experiment, Gualou Xiebai Banxia decoction combined with-Xuefu Zhuyu decoction were used to intervene the miniature swine phlegm and blood stasis type coronary heart disease model, in order to observe the effect of the combined prescription on the myocardial apoptosis and the expressions of Bcl-2, Bax, Caspase-3, Caspase-9 in the model. Totally 15 Chinese experimental miniature swine were adopted and randomly divided into the control group, the model group and the phlegm and stasis-treating group. The model group and the stasis-treating group were fed with high fat diets for two weeks, intervened with the coronary artery injury and then given drugs and high fat diets for eight weeks. The control group was fed with ordinary diets for 10 weeks, without the coronary artery injury. After the experiment, myocardia at the juncture of infracted areas were collected and made into formalin-fixed paraffin sections. The TDT-mediate dUTP nick end labeling (TUNEL) assay was used to detect the myocardial apoptosis. The immunohistochemistry (IHC) technique was applied to detect Bcl-2, Bax, Caspase-3, Caspase-9 levels in myocardial tissues. According to the findings, the apoptosis indexes (AI) for the control group, the model group and the phlegm and stasis-treating group were 0.92%, 27.68%, 17.28%, respectively. The AI of the phlegm and stasis-treating group was significantly lower than that of the model group (P < 0.01). Compared with the model group, the phlegm and stasis-treating group showed significantly higher Bcl-2 protein expression (P < 0.01) and lower Bax, Caspase-3 and Caspase-9 protein expressions (P < 0.01). In conclusion, Gualou Xiebai Banxia decoction combined with Xuefu Zhuyu decoction have a significant protective effect against the myocardial apoptosis in miniature swine phlegm and blood stasis type coronary heart disease model.
Animals ; Apoptosis ; drug effects ; Caspases ; metabolism ; Coronary Disease ; drug therapy ; Disease Models, Animal ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Male ; Medicine, Chinese Traditional ; Myocardium ; pathology ; Phytotherapy ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Swine ; Swine, Miniature ; bcl-2-Associated X Protein ; analysis

Objective To determine the protein expression of P14ARF,MDM2 and mutant type P53 (P53mt) in skin specimens of coal-burning-type of endemic arseniasis patients and to reveal the molecular mechanism of the disease.Methods Sixty skin specimens from 60 endemic arseniasis patients including 35 of skin lesions patients,19 of precancerous lesion and 6 of skin cancer and 9 normal skin specimens from non-cancer patients were studied.Expression of P14~,MDM2 and P53mt was evaluated by immunohistochemistry using corresponding monoclonal antibodies.Results There was significant difference in the positive rates of P14ARF,MDM2 and P53mt among the 4 groups(x2 =9.39,6.21,20.64,all P ＜ 0.05).The positive rates of P14ARF in precancerous lesion and skin cancer specimens were 46.1％ (6/19) and 33.3％ (2/6),respectively,which were significantly lower than that of the normal skin specimens [88.9％(8/9),all P ＜ 0.05].Decreased expression of P14ARF was correlated with the development of dermopathy (P ＜ 0.05).The positive rates of MDM2 and P53mt in skin lesions,precancerous lesion and skin cancer specimens were 54.2％ ( 19/35 ),63.2％ (10/19),66.7％ (4/6) and 25.7％(9/35),73.7％(14/19),83.3％(5/6),respectively,which were significantly higher than those of the control (0,0,all P＜ 0.05).The expression of MDM2 and P53mt increased with the development of dermopathy(all P ＜ 0.05).Conclusions P53mt protein in skin tissue of coal-burning-type of endemic arseniasis patients is over expressed.Abnormal expression of P14ARF and MDM2 may be one of the reasons lead to abnormal cell cycle control disorders and may play a role in the development of endemic arseniasis.

Objective To detect genetic polymorphism of myeloperoxidase (MPO) gene and catalase (CAT) gene and their activities, and to analyze their relationship with arsenic poisoning caused by coal-burning. Methods One hundred and thirty arsenic poisoning patients were chosen as case group in Jiaole Village, Xingren County, Guizhou Province(an endemic area). One hundred and forty healthy residents living in 13 km away were chosen as control group. Their blood was collected. Polymerase chain reaction-restriction fragment length polymorphism technique(PCR-RFLP) was used to detect polymorphism of MPO-463G/A and CAT-262C/T. Ultraviolet spectmphotometer method was used to detect myeloperoxidase activity. Chromatometry method was used to detect catalase activity. Results The genotype frequency of MPO-463G/A at GG, GA, AA site was 47.24%(60/127), 44.09%(56/127),8.67% (11/127) in case group and 42.34% (58/137),48.17% (66/137)1,9.49% (13/137) in control group, respectively. The difference between the two groups was not significant(χ2 = 0.642, P > 0.05). The genotype frequency of CAT-262C/T, at CC, CT, TT site was 65.60%(82/125),28.80%(36/125),5.60%(7/125) in case group and 76.51%(101/132), 18.94% (25/132) ,4.55% (6/132) in control group, respectively, without significant difference (χ2 =3.845, P>0.05). The relationship between polymorphism of MPO-463G/A and CAT-262C/T and the risk of arsenic poisoning was not found in this study(ORadj= 1.36, 95%CI: 0.74-2.50 for MPO; ORadj=1.35, 95%CI: 0.69-2.63 for CAT). The activities of MPO and CAT were (25.30±8.70)U/L and (2.80± 1.09)×103 U/L in case group, while (22.76±7.59)U/L and (3.90±1.01)×103U/L in control group with a significant difference(F=0.760 for MPO, F=0.855 for CAT, all P < 0.05). The genotype of MPO-463G/A and CAT-262C/T was not found to have relationship with the activities of MPO, CAT(F=1.312,2.822 for MPO; F= 0.151,0.036 for CAT, P>0.05). Conclusions Genetic polymorphism of MPO-463G/A and CAT-262C/T is not found to have relationship with arsenic poisoning. Arsenic can lead to the change of MPO and CAT activity, which, however, may not be affected by MPO-463G/A and CAT-262C/T polymorphism.

Objective To explore the influence of coal-arsenic exposure on human T cells proliferation and its mechanism.Methods Blood samples colleoted from individuals which lived in arsenism area of coal-burning type and non-arsenism area in Guizhou Province were divided into exposed group(17),mild(35),moderate(38) and severe arsenism group(19)and control group(35)according to Diagnosis Smndard for Endemic Arsenism (WS/T 211-2001).T cell stimulation index wag determined by methyl thiazolyl tetrazolium(MTT)colorimetric method.The intracellular Ca2+ exponential(IECa2+)in peripheral blood mononuclear cell(PBMC)was analyzed by Fho-3/AM dye and flow cytometry.DNA binding activity of actively T cells nuclear factor(NF-AT)in PBMC was evaluated by electrophoretie mobility shift assay(EMSA).Results Concanavalin A(ConA)stimulation decreased the T cells stimulation indexes in exposed group,mild,moderate and severe arsenism groups(1.315±0.962, 1.611±1.224,1.114±0.545,1.289±0.875)compared with control group(2.322±1.241),all the differences being statistically significant(P＜0.01).After stimulated by anti-CD3 monoclonal antibody(McAb),the T cells stimulation index in exposed group,mild,moderate and severe arsenism group(0.997±0.177,1.103±0.291,1.007±0.221, 0.957±0.205) were lower than that of control group(1.842±0.429,P ＜ 0.01 ). IECa2+ of PBMC after treated by anti-CD3 McAb in mild,moderate and severe arsenism group( 110.130±49.637,92.429±31.191,77.640± 35.372) were lower compared with control group(145.986±59.450,P ＜0.01 ). Moreover,IECa2+ in moderat and severe arsenism group were lower than exposed group(121.337±46.410,P ＜ 0.05). DNA binding activity of PBMC NF-AT in mild,moderate and severe arsenism group(1.354±0.446,1.290±0.291,1.159±0.411 ) were lowered than that of control group(1.722±0.291,P ＜ 0.01) and exposed group(1.611±0.294,P ＜ 0.05). Conclusions The coal-arsenic exposure can reduce the human T cells stimulation indexes,IECa2+ in PBMC and the DNA binding activity of NF-AT. It suggest that arsenic may suppress the proliferation ability of human T cells,which may be partly related to the influence of arsenic on T cell receptor(TCR)/CD3 signal transduetion pathway.

Objective To investigate the activation of T lymphocytes in human peripheral blood and the signaling molecules in protein kinase C/nuclear factor KB(PKC/NF-κB) pathway expressivity or activity changes in human peripheral blood mononuclear cells(PBMCs) exposed to coal-arsenic,to explore the role of PKC/NF-κB signal pathway in activation of T cells in human exposed to coal-arsenic. Methods Blood samples were collected from individuals who lived in arsenism area of coal-burning in Guizhou province, and were divided into asymptomatically exposed group (12),mild arsenism group (33),moderate arsenism group (34) and severe arscnism group (15) according to Diagnosis Standard for Endemic Arsenism (WS/T 211-2001). The individuals who lived in non-arsenism area were control group(27). The ratio of activated T ceils was analyzed by flow cytometry. DNA binding activity of NF-κB in PBMCs was evaluated by electrophoretic mobility shift assay(EMSA). The expression of PKCθ and phospho-PKCθ(pPKCθ) in PBMCs were detected with western blotting analysis. Results The ratio of activating T cells in asymptomatically exposed group[(21.76±15.31)%],mild arsenism group[(18.41±11.36)%],moderate arsenism group[(17.78±11.93)%]and severe arsenism group[(18.79±13.38)%]were all higher than that of control group[(3.19±2.12)%],the difference among all groups being statistically significant(F = 7.893,P < 0.05). DNA binding activity of PBMCs NF-κB in asymptomatically exposed group,mild arsenism group,moderate arsenism group and severe arsenism group(1.49±0.24,1.58±0.30,1.57±0.34,1.51±0.16) were higher than that of the control group(1.30±0.17),the difference being statistically sign/ficant(P < 0.05 or < 0.01). The expression of PBMCs pPKCθ in mild arsenism group,moderate arsenism group and severe arsenism group(0.64± 0.14,0.64±0.27,0.62±0.12) were all lower than that of the control group(0.93±0.20),the difference being statistically significant(P < 0.05). There were significant negative correlations between the expression of pPKCθ and the activity of NF-κB(r =-0.565,P < 0.01). There were significant positive correlations between the activity of NF-κB and the ratio of activating T cells(r = 0.546,P < 0.01). Conclusion Coal-arsenic enhances the DNA binding activity of NF-κB,reduces the expression of PBMCs pPKCθ in human PBMCs and up-regulates the activity of T cells. It suggests that the PKC/NF-κB signal might be one of transduction pathway via activating of T cells by coal-arsenic.

0.05).2.Neonates umbilical serum leptin concentration was positively correlated with body mass index(r=0.520 P