Journal Detail Information

Cite

DNA Sequence Analysis and Expression of the Recombinant Plasmid pBX1 from Borrelia Burgdorferi B31 Strain

Yong'en XIE ; Lang BAO ; Changhua HU ; Xuemin LI ; Wei CHEN

Journal of Sichuan University (Medical Sciences).2001;32(2):163-166. doi:10.3969/j.issn.1672-173X.2001.02.002

Objective　This study was to provide the target antigen for the development of a Lyme disease vaccine and serodiagnosis reagent.Methods　We used the automatic DNA sequencing machine (Model 377) to detect the nucleotide sequence of the inserted part of the recombinant plasmid pBX1 from Borrelia burgdorferi B31 strain. The restriction enzyme map of the inserted part of pBX1 was analysed by using computer software. The expressed product of pBX1 in E.coli XLI-Blue MRF′ was analysed by using SDS-PAGE and western-blotting. Results　①DNA sequencing showed that pBX1 contained a 477bp inserted gene fragment,and when it was compared with the published sequence of the specific region of the gene of the 83kd antigen protein from Borrelia burgdorferi B31 strain,only one amino acid codon was different.②The restriction enzyme map of the inserted part of pBX1 was successfully constructed.③The recombinant plasmid pBX1 expressed a 29kd fusion protein in E.coli XL1-Blue MRF′after induced with IPTG.The recombinant fusion protein could be recongnized by rabbit polyclonal antiserum against Borrelia burgdorferi B31 strain. Conclusion　A recombinant plasmid which contains the gene fragment encoding the specific region of the 83kd antigen protein from Borrelia burgdorferi B31 strain has been successfully constructed.The recombinant plasmid can stably express 29kd fusion protein in E.coli XL1-Blue MRF′.These results could serve as a base of further studies on the usefulness of the fusion protein in serodiagnosis and vaccine for Lyme disease.

Cite

The HVR Genotypes and Their Relationship with the Resistance of Methicillin-Resistant Staphylococci

Fang LIAO ; Xinjian FAN ; Xiaoju LÜ ; Ping FENG

Journal of Sichuan University (Medical Sciences).2001;32(2):167-171. doi:10.3969/j.issn.1672-173X.2001.02.003

Objective　To investigate the HVR-PCR genotype of methicillin-resistant Staphylococci in local hospitals and compare it with the antibiograms, with aview to selecting effective antibacterial agents, moreover, to discuss preliminarily its role in molecular epidemiology. Methods　 The minimal inhibitory concentrations(MICs) of 86 MRSA, 10 MRSE(Mcr S.epidemidis), 5 MSSE(Mcs S.epidemidis), 8 MRSH(Mcr S. haemolyticus) and 5 MSSH(Mcs S. haemolyticus) clinical isolates collected from 4 local hospitals were tested by serial two-fold agar dilution method; their DNA were extracted by moved basic lytic method, whose polymerase chain reaction(PCR) products amplified, based on the size of mec-associated hypervariable region(HVR) were analyzed by PAG vertical and agarose gel electrophoresis. Results 　 MRSA, MRSE and MRSH were grouped into 4, 3 and 2 HVR genotypes respectively according to the size of the PCR products. The PCR products amplified from 9 of 10 MRSE isolates were the same as the products amplified from MRSA isolates. MRSA strains in this study were mainly HVR genotypes A and D, which accounted for 52.32% and 39.53%； Genotypes B and C were the most multi-drug resistant, but genotype D was multi-sensitive. The Ⅰ genotype of MRSE was multi-drug resistant, but its genotype Ⅲ was multi-drug sensitive. The genotype a of MRSH was more resistant than genotype b. Conclusion These results suggest that HVR-PCR genotype method is an easy and fast method for epidemiological investigation of nosocomial infections caused by MRSA， and it is helpful for clinical selection of antibacterial agents. This method can compare the mec determinants of MRSA and Mcr CNSt isolates and hence to search for the origin of the mec determinant.

Cite

Construction of Recombinant Plasmids Containing Genes of HBsAg and Their Expression in the Eukaryotic Cells

Shan QIN ; Hong TANG ; Liansan ZHAO ; Li LIU ; Yong LIN

Journal of Sichuan University (Medical Sciences).2001;32(2):172-174. doi:10.3969/j.issn.1672-173X.2001.02.004

Objective　To construct recombinant plasmids expressing L,M,S and pre-S1-S protein of HBsAg. Methods　Amplifying segments of S, pre-S2-S, pre-S1-pre-S2-S genes of HBV by PCR and amplifying segment of pre-S1-S by overlap extension PCR; inserting the segments into Rc/CMV and pSG5UTPL/Flag plasmids respectively and exploring their expressions by Western-Blot hybridization，identifying the inserting segments by sequencing.Results　The sequences of the inserted segments were the same as the genes of S, pre-S2-S, pre-S1-pre-S2-S and pre-S1-S and the results of Western-Blot hybridization were positive for the aimed proteins. Conclusion　We have gained 8 recombinant plasmids expressing S, M, L and pre-S1-S proteins with high efficacy.

Cite

Apolipoprotein C Ⅲ Gene SstⅠ Polymorphism in Patients with Endogenous Hypertriglyceridemia in Chinese Population

Rui LIU ; Huai BAI ; Yu LIU ; Minghui HUANG ; Bingwen LIU

Journal of Sichuan University (Medical Sciences).2001;32(2):175-178,190. doi:10.3969/j.issn.1672-173X.2001.02.005

Objective　To investigate the apoCⅢ gene Sst Ⅰ polymorphism and its relationship with serum lipids and apolipoproteins (apo) levels in patients with endogenous hypertriglyceridemia in Chinese population. Methods　The genotype and allele frequency of apoCⅢ gene Sst Ⅰ polymorphism was assayed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Serum lipids were measured by enzymatic kits and apolipoproteins AⅠ, AⅡ, B100, CⅡ, CⅢ and E were measured by RID kits developed by Apolipoprotein Research Unit of WCUMS in 176 HTG patients whose fasting serum TG levels were ≥2.26 mmol/L and in 199 healthy subjects whose fasting serum TG levels were <1.82 mmol/L and TC levels <6.2 mmol/L from a population of Chinese Han nationality in Chengdu area. Results　In both HTG group and control group, S1 allele was the major allele and homozygous S1S1 genotype was the most frequent one. The frequency of S2 allele was significantly higher than that reported in Caucasians (0.289 vs 0.06～0.16, P<0.05). No differences were found in apoCⅢ gene Sst Ⅰ polymophism in the HTG group when compared with the control group (0.287 vs 0.289, P>0.05). The genotype of S2S2 was not associated with higher TG and apolipoproteins levels when compared with the genotypes of S1S1 and S1S2 (P>0.05). Conclusion　 These results suggest that the Sst Ⅰ polymorphism of the apoCⅢ gene was not associated with endogenoushypertriglyceridemica in Chinese population.

Cite

The Protein Expression of p53 and bcl-2 in Laryngeal Squamous Cell Carcinoma

Shixi LIU ; Siquan TANG ; Yinghe LIN ; Chuanyu LIANG

Journal of Sichuan University (Medical Sciences).2001;32(2):191-193. doi:10.3969/j.issn.1672-173X.2001.02.009

Objective　 To detect the relationship of p53 and bcl-2 gene to laryngeal carcinoma. Methods　p53 and bcl-2 protein expressions were examined in the specimens of 60 laryngeal carcinomas and 8 vocal cord polyps by immunohistochemistry method. Results　 Overexpressions of p53 and bcl-2 were found in the laryngeal carcinoma specimens; the positive rates of p53 and bcl-2 protein expression were 61.7%(37/60) and 43.3%(26/60), respectively. Nagative results were observed in all the vocal cord polyp specimens. The positive rates of p53 and bcl-2 expression were related to the histological grade and cervical lymphatic metastasis of laryngeal carcinoma but not related to clinical stage and tumor types. Conclusion　 The results of this study support the hypothesis that p53 and bcl-2 gene may be involved in the apoptosis of laryngeal carcinoma and they may play an important role in the pathogenesis of laryngeal cancer.

Cite

Adenovirus-mediated Rb Gene Transfect for Head and Neck Cancer

Xiaoqing ZHANG ; Shixi LIU ; Chuanyu LIANG ; Huan YANG

Journal of Sichuan University (Medical Sciences).2001;32(2):194-195,207. doi:10.3969/j.issn.1672-173X.2001.02.010

Objective　To evaluate the efficacy of Rb gene therapy in head and neck carcinoma. Methods　We investigated the adenovirus-mediated Rb (Ad-Rb) gene transfect into human oral squamous cell carcinoma cell line (012) in both in vitro and in vivo models. Results　The cells growth curve for 012 tumor cells transfect with Ad-Rb became lower after two days transfection and declined slightly by six days. Tumor growth in nude mice was also significantly retarded following Ad-Rb injection when compared with controls. Ad-Rb expression was identified by Western blotting in those tumors injected with Ad-Rb. Conclusion　These results implicated that the recombinant Rb gene adenovirus vector could efficiently transfect and inhibit oral squmous cell carcinoma. It might be an important candidate for gene therapy in head and neck cancer.

Cite

The Significance of Nuclear Factor κBp65 (NFκBp65) Expression on the Vascular Endothelial Cells of Rectum Adenocarcinoma of Human

Yao CHEN ; Ruixiang LI ; Ruohan WANG ; Zhiyu LIU

Journal of Sichuan University (Medical Sciences).2001;32(2):196-199. doi:10.3969/j.issn.1672-173X.2001.02.011

Objective　This study inquired into the mechanism of cancer vascular metastasis. Methods　The immunohistochemical method was adopted in determining the expression of intercellular adhesion molecule-1(ICAM-1) and nuclear factor κB (NFκBp65) on the peritumoral rectum tissues and metastatic lymph nodes of 8 patients and on the rectum tissues and lymph nodes of 5 normal human subjects. Also, the method of in situ hybridization was employed in detecting the binding site of NFκBp65 on vascular endothelial cells. Results　It was found that the proteins of ICAM-1 and NFκBp65 were expressed on the vascular endothelial cells of the rectum adenocarcinoma patients, and there was a NFκB binding consensus sequences on ICAM-1 promoter in the vascular endothelial cells of the rectum adenocarcinoma patients. When DIG-AKP labeled 38-bp oligonucleotide probe was used, there was no expression of ICAM-1 and NFκBp65 on the vascular endothelial cells of the normal human lymph node and rectum tissues. Conclusion　The above data suggest that the activation of ICAM-1 promoter may critically depend on NFκBp65 homodimers or heterodimers binding to a variant κB site on the vascular endothelial cells of human rectum adencarcinoma. These may indicate the potential roles of NFκB in cancer metastasis, thus giving clues to the development of a novel anti-metastasis strategy.

Cite

Alterations in the Level of Calcitonin Gene Related Peptide and Endothelin-1 in the Cirrhotic Rat Heart

Hua FU ; Li YANG ; Xiaojing LIU ; Hongde HU ; Ou QIANG ; Minghui HUANG

Journal of Sichuan University (Medical Sciences).2001;32(2):200-201,221. doi:10.3969/j.issn.1672-173X.2001.02.012

Objective　 To investigate the role of calcitonin gene related peptide(CGRP) and endothelin-1(ET-1) in cirrhotic cardiomyopathy (CCM). Methods　We measured the level of CGRP and ET-1 in the samples of rat heart collected from 15 liver cirrhosis rats and 15 controls by using radio immunoassay. Results　 The data showed that the levels of CGRP (74.2130±10.3776pg/mg protein)and ET-1 level(1.4780±0.9235pg/mg protein) were significantly higher in the cirrhotic rat hearts than those in controls (P<0.05). The increase of ET-1 in the cirrhotic rat hearts was closely associated with the severity of liver cirrhosis (P=0.004); whereas no significant association was seen between the CGRP concentration and the severity of liver cirrhosis (P=0.307). Conclusion　We infer that the increasing of CGRP level in the cirrhotic rat heart may be a protective or antagonistic reaction to ET-1 or other pathogenic factors for cardiac dysfunction. The disturbance of the balance between CGRP and ET-1 in the liver cirrhosis rat hearts may contribute to the pathologic process of CCM.

Cite

TGFβ1 and ET-1 Expression in the Peripheral Blood of Patients with Cirrhosis

Li YANG ; Xiaojing LIU ; Hua FU ; Ou QIANG ; Minghui HUANG

Journal of Sichuan University (Medical Sciences).2001;32(2):202-203,212. doi:10.3969/j.issn.1672-173X.2001.02.013

Objective　The aims of this study were to determine ① the variation of blood TGFβ1 and ET-1 in patients with liver cirrhosis; ② the relationship of the blood TGFβ1 and ET-1 with Child degree of liver function and the severity of esophageal varices. Methods　Peripheral blood samples were obtained from 72 patients and 24 normal controls.The blood levels of TGFβ1 and ET-1 were measured by using a standard bioassay and the radioimmunoassay.Results　TGFβ1 level （11.77±1.32 ng/ml）and ET-1 level（78.37±17.54 pg/ml） were significantly higher in the patients than those in controls(P<0.05). The increase of the ET-1 in the patients is closely associated with Child degree of liver function (r=0.94) and the severity of esophageal varices(r=0.87). TGFβ1 concentration is not associated with Child degree of liver function (r=0.11) and the severity of esophageal varices (r=0.03) . Conclusion　TGFβ1 and ET-1 may play an important role in the pathophysiologic process of cirrhosis. ET-1 concentration may reflect portal hypertension development.

Cite

Investigation of the Effects of Various Factors Affecting Apoptosis of Chronic Lymphocytic Leukemic Cells Mediated by P2Z Receptors

Liming PENG ; Qin ZHANG ; Hong JIANG ; Bradely J CHRISTOPHER ; Wiley S JAMES

Journal of Sichuan University (Medical Sciences).2001;32(2):208-212. doi:10.3969/j.issn.1672-173X.2001.02.015

Objective　To explore the effects of various factors affecting apoptosis of chronic lymphocytic leukemia (CLL) cells mediated by P2Z purinoceptor. Methods　In vitro exposure of CLL cells with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of 1.0 mmol/L Mg2+ or other bivalent cations, 150 mmol/L choline in media, 1,2,4 mmol/L EDTA or EGTA, or under different temperature. ATP-induced apoptosis was detected by agarose gel electrophoresis, TdT assay and flow cytometric analysis (FCA). Results　 The results showed that ① Extracellular Mg2+ or Ca2+ stimulated ATP-induced DNA fragmentation in a dose-dependent manner, and the compatible evidence was provided by the inhibition of ATP-induced DNA fragmentation in the presence of EGTA or EDTA; ② ATP-induced DNA fragmentation was completely inhibited by 1.0 mmol/L Zn2+; ③ ATP-induced DNA breaks were not affected by Ba2+, Sr2+, Co2+ when they were substituted for extracellular Mg2+ or Ca2+; ④ Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); ⑤ ATP-induced DNA fragmentation was completely obliterated when the temperature was lower than 10℃. Conclusion　These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.

DISPLAY OPTIONS

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share

Cite

Share