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With the sequence of the vasoactive intestinal peptiepeptide (VIP) from humans and according to the condon bias of Pichia pastoris, we designed PCR primers of VIP and obtained the sequence of VIP by SOE-PCR. Then VIP gene was cloned into Pichia pastoris secretory expression vector and the cell secretary system GS115-pPICZαA-vip was constructed. The recombinant strain was induced by methanol for 96 hours, and we collected the supernatant and identified the VIP by mass spectrometry. The molecular weight of VIP was consistent with theoretical molecular weight. The final result showed that the target peptide VIP was successfully expressed. The experimental investigations of agarose gel diffusion revealed that the recombinant expression modified VIP had relatively strong antibacterial activity to E. coli ATCC25922 and S. aureus ATCC25923. The minimal inhibitory concentration (MIC) of VIP to E. coli ATCC25922 and S. aureus ATCC25923 was 8 mmol/L and 16 mmol/L. Further cytotoxicity and hemolytic experiments indicated that recombinant VIP was non-toxic to normal cells NCM460 and IPEC-J2, had little hemolysis activity to SD rat erythrocytes. Meanwhile, by transmission electron microscopy, we found that VIP mainly inhibited bacteria by disrupting the cell membrane. These experiments established a useful system for further studies, application and mass production of antimicrobial peptide VIP.
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OBJECTIVE:To systematically evaluate the effectiveness and safety of escitalopram and duloxetine in the treatment of depression, and provide evidence-based reference for clinical treatment. METHODS:Retrieved from PubMed, Wanfang database,VIP,CNKI and CBM,randomized controlled trials (RCTs) about escitalopram (trial group) and duloxetine (control group) in the treatment of depression were collected. Meta-analysis was conducted by using Rev Man 5.3 software after data extraction and quality evaluation according to bias risk assessment tool recommended by system evaluator manual 5.3. RESULTS:Finally 25 RCTs were included,involving 2621 patients. The results of Meta-analysis showed that there was no statistical significance in total response rate between 2 groups after 1,2,4,6,8 weeks of treatment or cure rate between 2 groupsafter 4,6,8 weeks of treatment (P>0.05). There was no statistical significance in total response rate [RR=0.96,95％CI(0.88, 1.05),P=0.42] or cure rate [RR=0.91,95％CI(0.78,1.06),P=0.24] of female patients,as well as total response rate [RR=0.96, 95％CI(0.84,1.11),P=0.61] or cure rate [RR=0.90,95％CI(0.54,1.49),P=0.69] of elderly patients between 2 groups. The incidence of constipation [RR=0.59,95％CI (0.42,0.81),P=0.001],dry mouth [RR=0.65,95％CI(0.51,0.82),P=0.0004], nausea [RR=0.68,95％CI(0.56,0.83),P=0.0002] and decreased appetite [RR=0.74,95％CI(0.55,0.99),P=0.04] in trial group were significantly lower than control group,with statistical significance. CONCLUSIONS:The effectiveness of escitalopram is similar to duloxetine in the treatment of depression,but the safety of escitalopram is better than duloxetine.
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Objective To investigate the expression of androgen receptor(AR),ATAD2 in hepatocellular carcinoma(HCC) and the correlations with clinicopathological features,and the role of DHT/AR and ATAD2 in proliferation of HCC cells.Methods The samples of 75 patients with HCC in the First Affiliated Hospital of China Medical University from February 2012 to December 2012 were collected.LM3 and Huh7 cells were divided into control group,DHT group,DHT + CDX (bicalutamide) group and CDX group;and also divided into Ri-ATAD2 group (adding interference fragments) and Ri-C group (adding control vector sequence).Immunohistochemistry was used to detect the expression of AR and ATAD2,and to analyze the correlations between clinical features and survival of patients.Real-time PCR and Western Blot were used to detect the expression of AR and ATAD2,and CCK-8 was used to detect cell proliferation.Results HCC patient samples were grouped according to AR and ATAD2 expression.Compared with low AR expression group (n =31),the ratio of tumor ＜5 cm in high expression group (n =44) was higher,and the ratio of TNM stage Ⅰ + Ⅱ was lower.Compared with low ATAD2 expression group (n=35),the ratio of metastasis and tumor differentiation grade Ⅲ + Ⅳ was higher in high expression group (n=40),and the difference was statistically significant (P ＜ 0.05).The overall survival rate of patients with high expression of ATAD2 was lower than other patients,and the differences were statistically significant (P＜0.05).Multivariate Cox regression analysis showed that ATAD2 expression (HR=1.935,95％ CI:1.066～3.515) and metastasis (HR=2.212,95％ CI:1.059～4.619) were independent predictors of poor prognosis.Compared with LO2 cells,the mRNA and protein level of AR and ATAD2 in LM3 and Huh7 cells were significantly higher,and the differences were statistically significant (P＜0.05).And the proliferation rate of HCC cells increased significantly after 48 and 72 hours compared with the control group,and the differences were statistically significant (P＜0.05).After adding CDX,the proliferation of LM3 and Huh7 induced by DHT was inhibited.DHT enhanced the expression of ATAD2,while CDX inhibited the expression of ATAD2.The expression of ATAD2 protein decreased when LM3 and Huh7 cells were interfered.Compared with Ri-C group,the proliferation of HCC cells in Ri-ATAD2 group decreased significantly after the DHT treatment 48 and 72 hours,and the difference was statistically significant (P＜0.05).Conclusions DHT/AR promoted the proliferation of HCC cells by inducing ATAD2 expression.Modulating ATAD2 expression may be the potential mechanism of DHT/AR in HCC proliferation.
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Objective To investigate the impact of eotaxin-3 gene polymorphisms on the clinical effect of inhaled corticosteroids (ICS) to provide clinical basis for eotaxin-3 as the target spot for treating bronchial asthma.Methods One hundred and ninety-six cases of asthma and 196 cases as controls were selected from the outpatients and inpatients in our hospital.Peripheral blood samples were collected from the asthma patients and normal controls.PCR-RFLP was adopted to detect the genotypes of eotaxin-3 +2497T＞G and-+-77C＞T.The response of ICS treatment and the change situation of ACT scores were compared among asthmatic patients with various genotypes.Results Peripheral blood eosinophil(EOS) counts,EOS proportion and total IgE in the patients with TG genotype at+2497 locus were significantly decreased compared with those in the patients with TT genotype,the difference was statistically significant(P＜0.05).The level of PD20 in asthmatic patients with TG genotype was significantly higher than that in the patients with TT genotype,the difference was statistically significant[(0.07-±-0.03)mg vs.(0.03 ± 0.01)mg,t=2.45,P=0.048];whereas the above indicators had no statistical difference among 3 kinds of +-77 genotypes.During ICS treatment process in the patients with TT genotype at +-2497 locus,the FEV1％,PD20 value and ACT scores were significantly improved compared with those in the patients with TG genotype,the difference was statistically significant(P＜0.01).Conclusion The asthmatic patients with TT genotype at +-2497 locus were more sensitive to ICS treatment,regular ICS treatment can significantly improve the lung function and clinical symptom score in these patients.
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Objective To investigate the impact of eotaxin-3 gene polymorphisms on the clinical effect of inhaled corticosteroids (ICS) to provide clinical basis for eotaxin-3 as the target spot for treating bronchial asthma.Methods One hundred and ninety-six cases of asthma and 196 cases as controls were selected from the outpatients and inpatients in our hospital.Peripheral blood samples were collected from the asthma patients and normal controls.PCR-RFLP was adopted to detect the genotypes of eotaxin-3 +2497T＞G and-+-77C＞T.The response of ICS treatment and the change situation of ACT scores were compared among asthmatic patients with various genotypes.Results Peripheral blood eosinophil(EOS) counts,EOS proportion and total IgE in the patients with TG genotype at+2497 locus were significantly decreased compared with those in the patients with TT genotype,the difference was statistically significant(P＜0.05).The level of PD20 in asthmatic patients with TG genotype was significantly higher than that in the patients with TT genotype,the difference was statistically significant[(0.07-±-0.03)mg vs.(0.03 ± 0.01)mg,t=2.45,P=0.048];whereas the above indicators had no statistical difference among 3 kinds of +-77 genotypes.During ICS treatment process in the patients with TT genotype at +-2497 locus,the FEV1％,PD20 value and ACT scores were significantly improved compared with those in the patients with TG genotype,the difference was statistically significant(P＜0.01).Conclusion The asthmatic patients with TT genotype at +-2497 locus were more sensitive to ICS treatment,regular ICS treatment can significantly improve the lung function and clinical symptom score in these patients.
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Objective To explore a new method which could detect the misplacement of peripherally inserted central catheter (PICC).Methods The action of coughing was applied to 252 patients and observing the changes in dripping speed.Results The accuracy rate of this new method was 100.00％ (252/252).The success rate of first PICC in these patients was also 100.00％ (252/252).Conclusions It suggests that the new method Should be effective and convenient in the assessment of PICC misplacement and it can be applied to detect the misplacement occurred in and after the process of PICC.
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The recurrence rate of non -small cell lung cancer ( NSCLC) patients after radiotherapy and chemotherapy have been increasing .The therapy scheme consists of reirradiation、chemotherapy、and chemoradio-therapy,with the purpose of improving the local control and prolonging the survival time .Reirradiation is feasible for locally recurrence of non -small-cell lung cancer patients , treatment is security and could better improve quality of life in patients .The majority of patients are tolerable and have better short -term efficacy , No severe short term radiation induced injury is observed .But the long term radiation induced injury and long term efficacy need further investigation .In the present paper ,we review the roles of reirradiation for locally recurrence of non -small-cell lung cancer patients after radiotherapy and chemotherapy and the progress in clinical research .
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Objective To discuss comparison of different analgesic solution to the immune status and serum levels of inflammatory cytokines of the patients with breast cancer after surgery.Methods The patients of experimental group and control group were given Dizocine and fentanyl to analgesic respectively perioperative experimental.Then analysis and comparison the immune status and serum levels of the two groups.Results The T lymphocyte subsets ( The level of CD3 +、CD4 +and the ratio CD4 +/CD8 +) and the level fo NK cells of the exprimental group patients intraoperative and postoperative 1 d were significantly higer than the control group.The anti-inflammatory factor in the serum of the experimental group patients intraoperative and postoperative 1d was significantly higer than the control group.The proinflammatory factor in the serum of the experimental group patients intraoperative and postoperative 1 d was significantly lower than the control group.Conclusion Dizocine can significantly improve the serum inflammatory factors of the patients with brest cancer after surgery,and can adjust the patient’s immune status.
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Objective To analyze the complications ofventriculoperitoneal shunt in the treatment of pediatric hydrocephalus,and improve operative method,in order to reduce postoperative complications and improve the clinical curative effect.Methods A total of 39 infants with hydrocephalus who underwent adjustable shunt were retrospectively analyzed,including surgical methods,results,and complications,and put forward counter measures.Results Thirty cases of postoperative clinical symptoms obviously improved compared with preoperative,head CT review were clearly seen with ventricle retraction.Nine cases suffered with complications after operation.Obstruction of shunt tube was found in 4 cases,shunt exposed in 3 cases,and postoperative infection in 2 cases.Conclusions Ventriculoperitoneal shunt complications related to surgery itself.Improved surgical techniques,and taking appropriate treatment measures can effectively reduce the incidence of complications.
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Objective To explore the relationship between Helicobacter pylori (HP) infection and gastric carcinogenesis,and to investigate its mechanism.Methods Totally 333 elderly patients with different degrees of gastric mucosal lesions in our hospital were selected.Patients were divided into 4 groups:chronic superficial gastritis group (n=86),chronic atrophic gastritis group (n=92),gastric ulcer group (n=80) and gastric cancer group (n=75).HP infection in patients were detected by paraffin-embedded tissue sections and ELISA.The gene expressions of c-myc,p16 and p53 were detected by immunohistochemical SP method,and the results were analyzed.Results HP infection positive rate was significantly higher in gastric cancer group than those in the chronic superficial gastritis group and chronic atrophic gastritis group (both P＜ 0.05).There were no significant differences in the expression rates of c-myc,p16 and p53 between HP positive and negative patients in chronic superficial gastritis group (all P＞0.05).There were differences in the expression rates of cmyc and p53 (both P＜0.05),while the expression rate of p16 had no significant difference between HP positive and negative patients in chronic atrophic gastritis group (P ＞ 0.05).There were differences in the expression rates of c-myc,p16 and p53 between HP positive and negative patients in gastric ulcer group and gastric cancer group (all P ＜ 0.05).Conclusions There is a certain correlation between Helicobacter pylori infection and gastric carcinogenesis.It is important to take anti-inflammatory treatment timely and actively to prevent gastric cancer in patients with HP infection in gastric mucosal lesions.
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Objective To evaluate the biological safety of 125I-filled carbon nanotubes covered with metallic esophageal stent with regard to the normal esophagus before clinical application.Methods 125I-filled carbon nanotubes covered with metallic esophageal stent was prepared.Eighteen of New Zealand rabbits were randomly divided into three groups with 6 rabbits in each group.Three groups of stents,non-radioactive,low radio-activity ( 3.7 - 5.6 MBq),and high activity ( 11.1 - 13.0 MBq ) were placed in the midpiece of esophagus of rabbits.Esophagus opacification and three-diamensions DSA were performed at 0.5 h,7,14 and 30 d after insertion of the stents,respectively.The rabbits were killed at 30d after insertion of the stents,and histologic examinations of the esophageal walls were performed.Results In non-radioactive and low activity groups,1 of 6 rabbits died of wound infection at 1 and 3 d after surgery due to pulmonary infection,respectively.All specimens were obtained from 16 rabbits.Microscopically,in all rabbits of low activity and high activity groups,there were membrana mucosa necrotic and swell and breakage of the muscle fiber in esophageal submucosa and muscularis,submucosal inflammation,which were more severe in high activity group.In low activity group,one esophagus ectal membrane was involved,however,esophageal perforation did not develop.In high activity group,3 of 6rabbits esophageal perforation had developed,in which one esophagus mediastinum fistula developed,without inflammation.In non-radioactive group,it was almost normal in mucosa layer,a small amount of inflammatory cells were found in submucosal layer,and part of muscle fibers was fractured and no pathological changes of necrosis was found.Conclusions Radioactive 125I carbon nanotubes covered metallic stent with low activity(3.7 -5.6 MBq) can be used as intraluminal palliative brachytherapy,which is safe and effective.
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Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.
Subject(s)
Animals , Humans , Mice , 3T3 Cells , CD55 Antigens , Genetics , Pharmacology , DNA, Complementary , Genetics , Drug Synergism , Graft Rejection , Membrane Cofactor Protein , Genetics , Pharmacology , Recombinant Fusion Proteins , Genetics , Pharmacology , TransfectionABSTRACT
A full-length cDNA sequence encoding for the precursor of a venom peptide (named BmKCT) with homology to chlorotoxin has been isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The sequence of BmKCT is similar (68 % identities) to that of chlorotoxin isolated from Leiurus quinquestriatus quinquestriatus. To understand the biological function of BmKCT, this peptide was expressed using pGEX expression system and purified using GST affinity column and gel filtration.Whole cell patch-clamping recording showed that BmKCT could significantly inhibit chloride currents of gliomas cells, and the inhibitory effect was reversible. These results suggested that BmKCT might belong to the class of short chain toxins blocking the chloride ion channels.
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<p><b>OBJECTIVE</b>To investigate the effects of BmkTXK(beta), a newly purified 'long chain' peptide inhibitor of K(+) channels from the Chinese scorpion Buthus martensi Karsch (BmK), on the electrophysiological properties of isolated rabbit atrial myocytes.</p><p><b>METHODS</b>The standard whole-cell patch-clamp technique was used to study the effects of multiple concentrations of BmkTXK(beta) on potassium currents and action potentials.</p><p><b>RESULTS</b>BmkTXK(beta) produced concentration-dependent prolongation of action potential duration at 20%, 50%, and 90% repolarization (APD(20,50,90)) without any use-dependence. Meanwhile, it had no significant effect on RMP, APA, or V(max) (n = 9). At a dose of 1 micro mol/L, BmkTXK(beta) decreased I(to) by 41.4% (n = 10, P < 0.01) at a membrane potential of +50 mV [from (13.63 +/- 0.87) pA/pF to (7.98 +/- 0.78) pA/pF]. I(to) was reduced significantly with an IC(50) value of 1.82 micromol/L (95% confidence interval: 1.47 - 2.17 micro mol/L), in a clear concentration-dependent manner. BmkTXK(beta) blocked I(Ks) and I(Ks),tail with an IC(50) of 20.15 micromol/L and a 95% confidence interval of 16.93 - 23.37 micromol/L. At a concentration of 10 micromol/L, BmkTXK(beta) blocked both I(Ks) (mean reduction 37.3% +/- 4.2%, P < 0.01, n = 7) and I(Ks), tail (mean reduction 35.8% +/- 4.1%, P < 0.01, n = 7). At 0 mV, 10 micromol/L BmkTXK(beta) inhibited both I(Kr) (mean reduction 40.5% +/- 2.6%, P < 0.01, n = 6) and I(Kr), tail (mean reduction 42.3% +/- 2.9%, P < 0.01, n = 6). Blocking of I(Kr) by BmkTXK(beta) occurred in a concentration-dependent manner, with an IC(50) of 17.21 micromol/L (95% confidence interval: 14.76-19.66 micromol/L). An absence of effects on I(K1) was observed for BmkTXK(beta), with no change in reversal-potential (n = 6, P > 0.05).</p><p><b>CONCLUSIONS</b>BmkTXK(beta) exerts direct blocking effects on several potassium channels involved in cardiac repolarization, and has a strong effect on prolonging the repolarization of rabbit cardiomyocytes without reverse frequency dependence. This finding suggests that BmkTXK(beta) could be a promising class III drug for anti-arrhythmic therapy without the risk of proarrhythmia.</p>
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Animals , Rabbits , Action Potentials , Cells, Cultured , Heart Atria , Myocardium , Cell Biology , Patch-Clamp Techniques , Scorpion Venoms , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To elucidate the inhibitory effects of recombinant Chinese scorpion neurotoxin BmK IM on seizures induced by pentylenetetrazol (PTZ) and the possible mechanism.</p><p><b>METHODS</b>After purifying recombinant BmK IM from an E. coli cell line, its toxicity (both LD50 and minimum lethal dose) on rats was determined. BmK IM was then microinjected into the CA3 region of the right hippocampus and its ability to inhibit the effects of an intraperitoneal injection of PTZ was assessed. The effects of BmK IM on the electrophysiological properties of isolated CA3 pyramidal neurons were then studied using whole-cell patch clamp techniques.</p><p><b>RESULTS</b>BmK IM can significantly prolong the latent period of epileptic seizures, decrease the degree of seizures, and decrease the frequency of epileptiform discharges induced by PTZ. At the same time, 24h after injection of BmK IM into the hippocampal tissue, BmK IM significantly reduces the concentration of the neurotransmitter glutamate and alleviates PTZ-induced lesions in the hippocampus. Whole-cell patch clamp recordings indicate that BmK IM inhibits INa of rat hippocampal neurons in a dose-dependent manner. BmK IM significantly shifts the activation curve of INa in a positive direction, indicating that BmK IM enhances the threshold potential of INa.</p><p><b>CONCLUSIONS</b>BmK IM has significant anti-epileptic properties, and may prove useful as a drug in the therapy of epilepsy. The inhibitory effects of BmK IM on seizures caused by pentylenetetrazol might depend on reductions in the release of presynaptic glutamate via the blocking of Na+ channels.</p>
Subject(s)
Animals , Male , Rats , Glutamine , Bodily Secretions , Hippocampus , Microinjections , Pentylenetetrazole , Peptides , Therapeutic Uses , Rats, Sprague-Dawley , Recombinant Proteins , Therapeutic Uses , Scorpion Venoms , Therapeutic Uses , Seizures , Sodium ChannelsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of antisense glutamic acid decarboxylase (GAD(67)) oligodeoxynucleo-tide (ODN) on behavior, seizure threshold and EEG of hippocampus in the epileptic rats induced by pentylenetetrazol (PTZ).</p><p><b>METHODS</b>A model of chronic epilepsy in rats was established by PTZ. The inhibition of GAD(67) mRNA expression in hippocampus was selectively induced by antisense oligodeoxynucleotide of GAD(67). The effect of antisense GAD(67) ODN on behavior, seizure threshold and EEG recording of kindled rats was examined.</p><p><b>RESULTS</b>Antisense GAD(67) ODN could inhibit the expression of GAD(67) mRNA and the concentration of GABA. It also could significantly shorten the latencies of seizure and increase the level of seizure and the frequency of epileptiform discharges.</p><p><b>CONCLUSION</b>The gene of GAD(67) may be an anti-seizure gene, which might inhibit epileptiform discharge. The treatment of epilepsy by GAD(67) gene will have a bright future.</p>
Subject(s)
Animals , Male , Rats , Electroencephalography , Epilepsy , Glutamate Decarboxylase , Genetics , Pharmacology , Hippocampus , Isoenzymes , Genetics , Pharmacology , Kindling, Neurologic , Oligonucleotides, Antisense , Pharmacology , Pentylenetetrazole , gamma-Aminobutyric AcidABSTRACT
Objective:To construct recombinant expressing vector pcDNA3-DAF and to develop the NIH3T3 cell model expess human complement regulatory protein decay accelerating factor(DAF,CD55)stably after transfected.Methods:Human membrane complement regulatory protein(hCRP) DAF cDNA containing the full-length of encoding region was cloned into expressing vector pcDNA3.After identification by restriction enzyme digestion,PCR and sequencing,the recombinant plasmid was transfected into NIH3T3 cells with calcium phosphate-DNA precipitate method.A stably-transfected cell line was established by G418 selection.Extraneous gene integration was identified by PCR.Expression of DAF at both mRNA and protein levels was analyzed by RT-PCR,Western blot and indirect immunofluorescence microscopy.Results:The eukaryotic expression vector pcDNA3-DAF was successfully constructed and the DAF gene was transfected stably into NIH3T3 cells,a stably-transfected cell line was established and DAF was efficiently expressed on the surface of transfected NIH3T3 cells.Human DAF cDNA was integrated into NIH3T3 pcDNA3-DAF genomic DNA after continuous 30 times passages,indicating that NIH3T3 pcDNA3-DAF was stable cell line.Conclusion:The establishment of the stably-transfected cell line and the expression of the target gene provide a base for further studies on the function of the DAF and the cooperative fashion among different human complement regulatory proteins in alleviating the complement-mediated cytolysis.
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Objective To get a molluscicide from Reineckea carnea which is effective on Oncomelania hupensis but has low poison on fish. Methods Methyl soaking, column chromatography and HPLC were used for isolation of molluscicidal component from Reineckea carnea. Results The snail mortality with Reineckea carnea powder was 86.0% for 72 h immersion and 100.0% for 120 h at the concentration of 17.5 mg/L, but the fish mortality was 0 at the concentration of 130 mg/L, indicating that Reineckea carnea had different effects on the two kinds of animals . The fraction A was obtained from Reineckea carnea by methyl immersion, column chromatography. The snail mortality with A was 84.4% for 144 h at the concentration of 10 mg/L, indicating that A had high molluscicidal effect on Oncomelania hupensis. After A being isolated by HPLC, 99.98% RC-Ⅰ was obtained from it . RC-Ⅰ was determined by API2000 LC/MS and other spectrum, and it was deduced to be one of steroidal glycosides. Conclusion Reineckea carnea contains a high effective molluscicidal component.