Experiments were conducted to study the adsorption of Cd on two soil colloids (red soil and yellow- brown soil) and three variable-charge minerals (goethite, noncrystalline Fe oxide and kaolin) in the absence and pres...Experiments were conducted to study the adsorption of Cd on two soil colloids (red soil and yellow- brown soil) and three variable-charge minerals (goethite, noncrystalline Fe oxide and kaolin) in the absence and presence of rhizobia. The tested strain Rhizobium fredii C6, tolerant to 0.8 mmol L-1 Cd, was selected from 30 rhizobial strains. Results showed that the isotherms for the adsorption of Cd by examined soil colloids and minerals in the presence of rhizobia could be described by Langmuir equation. Within the range of the numbers of rhizobial cells studied, the amount of Cd adsorbed by each system increased with increasing rhizobial cells. Greater increases for the adsorption of Cd were found in red soil and kaolin systems. Rhizobia influence on the adsorption of Cd by examined soil colloids and minerals was different from that on the adsorption of Cu. The presence of rhizobia increased the adsorption sanity of soil colloids and minerals for Cd, particularly for the goethite and kaolin systems. The discrepancies in the influence of rhizobia on the adsorbability and affinity of selected soil colloids and minerals for Cd suggested the different interactions of rhizobia with various soil components. It is assumed that bacterial biomass plays an important role in controlling the mobility and bioavailability of Cd in soils with kaolinite and goethite as the major colloidal components, such as in variable-charge soil.展开更多
AIM: To investigate the inhibitory effects of endostatinvascular endothelial growth inhibitor (VEGI151) recombinant adenoviruses on neovascularization.METHODS: We used recombinant adenoviruses to treat human vascular ...AIM: To investigate the inhibitory effects of endostatinvascular endothelial growth inhibitor (VEGI151) recombinant adenoviruses on neovascularization.METHODS: We used recombinant adenoviruses to treat human vascular endothelial cell line ECV304, human hepatocellular carcinoma cell line HepG2, and murine fibroblast cell line L929, in order to study the chimeric gene expression in these cell lines. Chick choriallantic membrane(CAM) model, rabbit inflammatory corneal neovascularization(CNV) model, and liver cancer-bearing nude mice model were employed to investigate the negative biological effect of fusion molecules on neovascularization in vivo.RESULTS: Western blot showed that the molecular weight of fusion protein was about 41kD after infection of ECV304,HepG2 and L929 cells with supernatant of AdhENDO-VEGI151.The fusion protein showed a specific inhibitory effect on the proliferation of ECV304 cells, but no inhibitory effect on the growth of HepG2 and L929 cells (F=13112.13, P=0.0001). In the chick choriallantic membrane (CAM) assay, the expressed fusion protein significantly inhibited neovascularization. Rabbit inflammatory corneal neovascularization (CNV) induced by intrastromal sutures resulted in a uniform neovascular response. In this model, direct subconjunctival injection of AdhENDO-VEGI151 expressed the fusion protein in vivo and suppressed the development of CNV. Topical application of AdhENDO-VEGI151 led to a significant suppression of CNV (F=1413.11, P=0.0001),as compared with the control group of AdLacZ. Immunohistochemical staining showed the fusion protein dominantly expressed in corneal epithelium. Compared with the control group of AdLacZ (4075.9±1849.9 mm^3), the average tumor size of group AdhENDO-VEGI151 reduced in size (487.7±241.2 mm^3) (F=14.80, P=0.0085), with an inhibition rate of 88.03%. Immunohistochemical staining showed the adenoviruses carried the fusion gene expressed on liver cancer cell membrane. MVD decreased more significantly in treated mice (30.75±3.31%) than in AdLacZ control(50.25±8.65%) (F=17.72, P=0.0056) with an inhibition rate of 39%.CONCLUSION: Fusion protein expressed by recombinant adenoviruses has a significant inhibitory effect on neovascularization.展开更多
Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNsare produced following viral infections, but until recently, the mechanisms of viral recognition leading ...Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNsare produced following viral infections, but until recently, the mechanisms of viral recognition leading to IFN productionwere largely unknown. Toll like receptors (TLRs) have emerged as key transducers of type I IFN during viral infectionsby recognizing various viral components. Furthermore, much progress has been made in defining the signaling path-ways downstream of TLRs for type I IFN production. TLR7 and TLR9 have become apparent as universally importantin inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells. New intracellularviral pattern recognition receptors leading to type I IFN production have been identified. Many bacteria can also inducethe up-regulation of these cytokines. Interestingly, recent studies have found a detrimental effect on host cells if type IIFN is produced during infection with the intracellular gram-positive bacterial pathogen, Listeria monocytogenes. Thisreview will discuss the recent advances made in defining the signaling pathways leading to type I IFN production.展开更多
AIM: To investigate the interrelationship between Epstein-Barr virus (EBV)-encoded proteins and cell proliferation, apoptosis and apoptosis-related proteins in gastric carcinoma, and to explore their role in gastric c...AIM: To investigate the interrelationship between Epstein-Barr virus (EBV)-encoded proteins and cell proliferation, apoptosis and apoptosis-related proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis. METHODS: Tissues from 13 cases of EBV-associated gastric carcinoma (EBVaGC) and 45 cases of matched EBV-negative gastric carcinoma (EBVnGC) were collected, and then subjected to analysis for apoptotic index (AI) using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Nuclear cell proliferation-associated antigen ki-67 index (KI), bcl-2, and p53 expression were examined by immunohistochemistry.p53 mutation in exons 5-8 of 13 EBVaGC cases was determined by single-strand conformation polymorphism (SSCP) and DNA sequencing.RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (IMP) 1, immediately early gene BZLF1 and early genes BARF1 and BHRF1 in 13 EBVaGC cases. RESULTS: The percentage of AI, KI and p53 overexpression was significantly lower in the EBVaGC group than in the EBVnGC group. However, bcl-2 expression did not show significant difference between the two groups. p53 gene mutations were not found in 13 EBVaGCs. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the thirteen cases exhibited BZLF1 transcripts and two exhibited BHRF1 transcripts. BARF1 mRNA was detected in six cases. CONCLUSION: Lower AI and KI may reflect a low biological activity in EBVaGC. EBV infection is associated with p53 abnormal expression but not bcl-2 protein in EBVaGC. BZLF1,BARF1,and BHRF1 may play important roles in inhibiting cell apoptosis and tumorigenesis of EBVaGC through different pathways.展开更多
Hepatitis E virus (HEV) is an unclassified, small, nonenveloped RNA virus, as a causative agent of acute hepatitis E that is transmitted principally via the fecal-oral route. The virus can cause large water-born epide...Hepatitis E virus (HEV) is an unclassified, small, nonenveloped RNA virus, as a causative agent of acute hepatitis E that is transmitted principally via the fecal-oral route. The virus can cause large water-born epidemics of the disease and sporadic cases as well. Hepatitis E occurs predominantly in developing countries, usually affecting young adults, witha high fatality rate up to 15-20% in pregnant women.However, no effective treatment currently exists for hepatitis E, and the only cure is prevention. But so far there are no commercial vaccines for hepatitis E available in the world.Although at least four major genotypes of HEV have been identified to date, only one serotype of HEV is recognized.So there is a possibility to produce a broadly protective vaccine. Several studies for the development of an effective vaccine against hepatitis E are in progress and the bestcandidate at present for a hepatitis E vaccine is a recombinant HEV capsid antigen expressed in insect cells from abaculovirus vector. In this article, the recent advances of hepatitis E and the development of vaccine research forHEV including recombinant protein vaccine, DNA vaccine and the recombinant hepatitis E virus like particles (rHEVVLPs) are briefly reviewed.展开更多
AIM: To determine the genotypes and phylogeny of hepatitis B viruses (HBVs) in asymptomatic HBV carriers, and the prevalence of occult HBV infection in Long An County, Guangxi Zhuang Autonomous Region, an area with a ...AIM: To determine the genotypes and phylogeny of hepatitis B viruses (HBVs) in asymptomatic HBV carriers, and the prevalence of occult HBV infection in Long An County, Guangxi Zhuang Autonomous Region, an area with a high incidence of hepatocellular carcinoma. METHODS. A nested polymerase chain reaction (nPCR) was used for detection of HBV DNA in serum samples from 36 blood donors with asympmatic HBV infection, and in serum samples from 52 HBsAg negative family members of the children who did not receive hepatitis B vaccination in Long An County. PCR products were sequenced, and the genotype of each HBV sequence was determined by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases using the BLAST programme. Phylogenetic trees were constructed by the quartet maximum likelihood analysis using the TreePuzzle software. RESULTS: Twenty (55.56%) of 36 HBV asymptomatic carriers were positive for HBV DNA. They were all genotype C by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases. The full-length HBV DNA sequence isolated from the sample No. 624 contained 3215 bases. No interesting mutations were found in this isolate. The homology analysis showed that this strain was closer to the Vietnamese HBV genotype C strain, with a homology of 97%, compared its relation to the same genotype of HBV isolated in Shanghai. Six (11.5%) of the 52 HBsAg negative family members were positive for HBV DNA. A point mutation was found in the sample No. 37, resulting in the substitution of amino acid glycine to arginine in the “a” determinant. Other samples with positive HBV DNA did not have any unusual amino acid substitutions in or around the “a” determinant, and were attributed to the wild-type HBV. CONCLUSION: The HBVs isolated from asymptomatic carriers of Long An County were all identified as genotype C, and the prevalence of occult HBV infection in the population of the county is as high as 11.5%. It is suggested that genotype C and persistent occult HBV infection may play an important role in the development of HCC in the county.展开更多
AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine.METHODS: By nitrosoguanidine mutagenesis, five p...AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine.METHODS: By nitrosoguanidine mutagenesis, five pfluorophenylalanine (FP)-resistant mutants of Cglutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroGpheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pl-K and pTGAK, respectively. Then,they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays.RESULTS: Engineering strains of C.glutamicum (Tyr-)were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold,and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-Darabinoheptulosonate-7-phosphate synthase, respectively.CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.展开更多
AIM: To investigate the effect of interleukin-12 p40 gene (IL12E 3'-untranslated region polymorphism on the outcome of HCV infection.METHODS: A total of 133 patients who had been infected with HCV for 12-25 (18.2...AIM: To investigate the effect of interleukin-12 p40 gene (IL12E 3'-untranslated region polymorphism on the outcome of HCV infection.METHODS: A total of 133 patients who had been infected with HCV for 12-25 (18.2±3.8) years, were enrolled in this study. Liver biochemical tests were performed with an automated analyzer and HCV RNA was detected by fluorogenic quantitative polymerase chain reaction. B-mode ultrasound was used for liver examination. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) was used for the detection of IL12B (1188A/C) polymorphism.RESULTS: Self-limited infection was associated with AC genotype (OR = 3.48; P = 0.001) and persistent infection was associated with AA genotype (OR = 0.34; P = 0.014)at site 1188 of IL12B. In patients with persistent HCV infection, no significant differences were found regarding the age, gender, duration of infection and biochemical characteristics (P>0.05). According to B-mode ultrasound imaging and clinical diagnosis, patients with persistent infection were divided into groups based on the severity of infection. No significant differences were found in the frequency of IL-12 genotype (1188A/C) between different groups (P>0.05).CONCLUSION: The polymorphism of II12B (1188A/C)appears to have some influence on the outcome of HCV infection.展开更多
AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC ...AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level, METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (x2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLFl transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.展开更多
AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determ...AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of Hpyloriwere associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: Hpyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the Hpyloripositive patients, 75% (38/51) had Hpyloriin all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori，irrespective of the clinical diagnosis.展开更多
AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: ...AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.展开更多
AIM: To study the effect of co-expression of ppsA, pckA,aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strainfor L-phenylalanineoMETHODS: ppsA and pckA genes wer...AIM: To study the effect of co-expression of ppsA, pckA,aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strainfor L-phenylalanineoMETHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coil and Brevibactenurn flavum to generate constructs pJN2 and pJNS.pJN2 was generated by inserting ppsA and pCKA genes into vector pCZ; whereas pJN5 was obtained by introducing ppSA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes.The recombinant plasmids were then introduced into B.flavum by electroporation and the transformants were used for L-phenylalanine fermentation.RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably, pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly.CONCLUSION: Co-expression of ppsA and pckA. genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pcsA, aroG, pheA and tyrB of E. coil in B. flavum was a feasible approach to construct a strain for phenylalanine production.展开更多
AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice.METHODS: A eukaryotic expression vector of pcDNA3.1-...AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice.METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector,whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid.Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared.RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line.No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01).The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group.CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.展开更多
The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD...The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.展开更多
AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients.METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isola...AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients.METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively.RESULTS: About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA+ strains contained 2-4tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro.CONCLUSION: CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.展开更多
基金Project supported by the National Natural Science Foundation of China !(No. 49601011)the Natural Science Foundation of Hubei
文摘Experiments were conducted to study the adsorption of Cd on two soil colloids (red soil and yellow- brown soil) and three variable-charge minerals (goethite, noncrystalline Fe oxide and kaolin) in the absence and presence of rhizobia. The tested strain Rhizobium fredii C6, tolerant to 0.8 mmol L-1 Cd, was selected from 30 rhizobial strains. Results showed that the isotherms for the adsorption of Cd by examined soil colloids and minerals in the presence of rhizobia could be described by Langmuir equation. Within the range of the numbers of rhizobial cells studied, the amount of Cd adsorbed by each system increased with increasing rhizobial cells. Greater increases for the adsorption of Cd were found in red soil and kaolin systems. Rhizobia influence on the adsorption of Cd by examined soil colloids and minerals was different from that on the adsorption of Cu. The presence of rhizobia increased the adsorption sanity of soil colloids and minerals for Cd, particularly for the goethite and kaolin systems. The discrepancies in the influence of rhizobia on the adsorbability and affinity of selected soil colloids and minerals for Cd suggested the different interactions of rhizobia with various soil components. It is assumed that bacterial biomass plays an important role in controlling the mobility and bioavailability of Cd in soils with kaolinite and goethite as the major colloidal components, such as in variable-charge soil.
基金Supported by The National Natural Science Foundation of China,No.30170891 and No.39970819
文摘AIM: To investigate the inhibitory effects of endostatinvascular endothelial growth inhibitor (VEGI151) recombinant adenoviruses on neovascularization.METHODS: We used recombinant adenoviruses to treat human vascular endothelial cell line ECV304, human hepatocellular carcinoma cell line HepG2, and murine fibroblast cell line L929, in order to study the chimeric gene expression in these cell lines. Chick choriallantic membrane(CAM) model, rabbit inflammatory corneal neovascularization(CNV) model, and liver cancer-bearing nude mice model were employed to investigate the negative biological effect of fusion molecules on neovascularization in vivo.RESULTS: Western blot showed that the molecular weight of fusion protein was about 41kD after infection of ECV304,HepG2 and L929 cells with supernatant of AdhENDO-VEGI151.The fusion protein showed a specific inhibitory effect on the proliferation of ECV304 cells, but no inhibitory effect on the growth of HepG2 and L929 cells (F=13112.13, P=0.0001). In the chick choriallantic membrane (CAM) assay, the expressed fusion protein significantly inhibited neovascularization. Rabbit inflammatory corneal neovascularization (CNV) induced by intrastromal sutures resulted in a uniform neovascular response. In this model, direct subconjunctival injection of AdhENDO-VEGI151 expressed the fusion protein in vivo and suppressed the development of CNV. Topical application of AdhENDO-VEGI151 led to a significant suppression of CNV (F=1413.11, P=0.0001),as compared with the control group of AdLacZ. Immunohistochemical staining showed the fusion protein dominantly expressed in corneal epithelium. Compared with the control group of AdLacZ (4075.9±1849.9 mm^3), the average tumor size of group AdhENDO-VEGI151 reduced in size (487.7±241.2 mm^3) (F=14.80, P=0.0085), with an inhibition rate of 88.03%. Immunohistochemical staining showed the adenoviruses carried the fusion gene expressed on liver cancer cell membrane. MVD decreased more significantly in treated mice (30.75±3.31%) than in AdLacZ control(50.25±8.65%) (F=17.72, P=0.0056) with an inhibition rate of 39%.CONCLUSION: Fusion protein expressed by recombinant adenoviruses has a significant inhibitory effect on neovascularization.
基金A. K. Perry is supported by the Howard Hughes Medi-cal Institute predoctoral fellowship (Grant No. 59003787).Part of this work was also supported by National Insti-tutes of Health research grants RO1 CA87924, RO1AI056154, and R37 AI47868 to G. Cheng and from the MajorResearch Plan (30170461, 30430640) +1 种基金Natural ScienceFoundation of China, and the National Basic ResearchProgram of MOST (2002CB513001, 2001CB-510002)H. Tang. H. Tang is also a fellow of Outstanding YoungInvestigators of National Naturual Science Foundation ofChina (30025010).
文摘Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNsare produced following viral infections, but until recently, the mechanisms of viral recognition leading to IFN productionwere largely unknown. Toll like receptors (TLRs) have emerged as key transducers of type I IFN during viral infectionsby recognizing various viral components. Furthermore, much progress has been made in defining the signaling path-ways downstream of TLRs for type I IFN production. TLR7 and TLR9 have become apparent as universally importantin inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells. New intracellularviral pattern recognition receptors leading to type I IFN production have been identified. Many bacteria can also inducethe up-regulation of these cytokines. Interestingly, recent studies have found a detrimental effect on host cells if type IIFN is produced during infection with the intracellular gram-positive bacterial pathogen, Listeria monocytogenes. Thisreview will discuss the recent advances made in defining the signaling pathways leading to type I IFN production.
文摘AIM: To investigate the interrelationship between Epstein-Barr virus (EBV)-encoded proteins and cell proliferation, apoptosis and apoptosis-related proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis. METHODS: Tissues from 13 cases of EBV-associated gastric carcinoma (EBVaGC) and 45 cases of matched EBV-negative gastric carcinoma (EBVnGC) were collected, and then subjected to analysis for apoptotic index (AI) using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Nuclear cell proliferation-associated antigen ki-67 index (KI), bcl-2, and p53 expression were examined by immunohistochemistry.p53 mutation in exons 5-8 of 13 EBVaGC cases was determined by single-strand conformation polymorphism (SSCP) and DNA sequencing.RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (IMP) 1, immediately early gene BZLF1 and early genes BARF1 and BHRF1 in 13 EBVaGC cases. RESULTS: The percentage of AI, KI and p53 overexpression was significantly lower in the EBVaGC group than in the EBVnGC group. However, bcl-2 expression did not show significant difference between the two groups. p53 gene mutations were not found in 13 EBVaGCs. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the thirteen cases exhibited BZLF1 transcripts and two exhibited BHRF1 transcripts. BARF1 mRNA was detected in six cases. CONCLUSION: Lower AI and KI may reflect a low biological activity in EBVaGC. EBV infection is associated with p53 abnormal expression but not bcl-2 protein in EBVaGC. BZLF1,BARF1,and BHRF1 may play important roles in inhibiting cell apoptosis and tumorigenesis of EBVaGC through different pathways.
基金Supported by the National Major Projects of National Committee of Science and Technology (2502AA2Z3342),and the Beijing Municipal Committee of Science and Technology (H020920020190)
文摘Hepatitis E virus (HEV) is an unclassified, small, nonenveloped RNA virus, as a causative agent of acute hepatitis E that is transmitted principally via the fecal-oral route. The virus can cause large water-born epidemics of the disease and sporadic cases as well. Hepatitis E occurs predominantly in developing countries, usually affecting young adults, witha high fatality rate up to 15-20% in pregnant women.However, no effective treatment currently exists for hepatitis E, and the only cure is prevention. But so far there are no commercial vaccines for hepatitis E available in the world.Although at least four major genotypes of HEV have been identified to date, only one serotype of HEV is recognized.So there is a possibility to produce a broadly protective vaccine. Several studies for the development of an effective vaccine against hepatitis E are in progress and the bestcandidate at present for a hepatitis E vaccine is a recombinant HEV capsid antigen expressed in insect cells from abaculovirus vector. In this article, the recent advances of hepatitis E and the development of vaccine research forHEV including recombinant protein vaccine, DNA vaccine and the recombinant hepatitis E virus like particles (rHEVVLPs) are briefly reviewed.
基金Supported by Beijing Municipal Committee of Science and Technology,No.H020920020190
文摘AIM: To determine the genotypes and phylogeny of hepatitis B viruses (HBVs) in asymptomatic HBV carriers, and the prevalence of occult HBV infection in Long An County, Guangxi Zhuang Autonomous Region, an area with a high incidence of hepatocellular carcinoma. METHODS. A nested polymerase chain reaction (nPCR) was used for detection of HBV DNA in serum samples from 36 blood donors with asympmatic HBV infection, and in serum samples from 52 HBsAg negative family members of the children who did not receive hepatitis B vaccination in Long An County. PCR products were sequenced, and the genotype of each HBV sequence was determined by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases using the BLAST programme. Phylogenetic trees were constructed by the quartet maximum likelihood analysis using the TreePuzzle software. RESULTS: Twenty (55.56%) of 36 HBV asymptomatic carriers were positive for HBV DNA. They were all genotype C by comparison with sequences of known genotypes in the GenBank and EMBL nucleotide databases. The full-length HBV DNA sequence isolated from the sample No. 624 contained 3215 bases. No interesting mutations were found in this isolate. The homology analysis showed that this strain was closer to the Vietnamese HBV genotype C strain, with a homology of 97%, compared its relation to the same genotype of HBV isolated in Shanghai. Six (11.5%) of the 52 HBsAg negative family members were positive for HBV DNA. A point mutation was found in the sample No. 37, resulting in the substitution of amino acid glycine to arginine in the “a” determinant. Other samples with positive HBV DNA did not have any unusual amino acid substitutions in or around the “a” determinant, and were attributed to the wild-type HBV. CONCLUSION: The HBVs isolated from asymptomatic carriers of Long An County were all identified as genotype C, and the prevalence of occult HBV infection in the population of the county is as high as 11.5%. It is suggested that genotype C and persistent occult HBV infection may play an important role in the development of HCC in the county.
基金Supported by the National Natural Science Foundation of China,No.30070020
文摘AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine.METHODS: By nitrosoguanidine mutagenesis, five pfluorophenylalanine (FP)-resistant mutants of Cglutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroGpheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pl-K and pTGAK, respectively. Then,they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays.RESULTS: Engineering strains of C.glutamicum (Tyr-)were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold,and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-Darabinoheptulosonate-7-phosphate synthase, respectively.CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.
基金Supported by the National Key Technologies Research and Development Program of China during the 10th Five-Year Period,No.2001BA705B06
文摘AIM: To investigate the effect of interleukin-12 p40 gene (IL12E 3'-untranslated region polymorphism on the outcome of HCV infection.METHODS: A total of 133 patients who had been infected with HCV for 12-25 (18.2±3.8) years, were enrolled in this study. Liver biochemical tests were performed with an automated analyzer and HCV RNA was detected by fluorogenic quantitative polymerase chain reaction. B-mode ultrasound was used for liver examination. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) was used for the detection of IL12B (1188A/C) polymorphism.RESULTS: Self-limited infection was associated with AC genotype (OR = 3.48; P = 0.001) and persistent infection was associated with AA genotype (OR = 0.34; P = 0.014)at site 1188 of IL12B. In patients with persistent HCV infection, no significant differences were found regarding the age, gender, duration of infection and biochemical characteristics (P>0.05). According to B-mode ultrasound imaging and clinical diagnosis, patients with persistent infection were divided into groups based on the severity of infection. No significant differences were found in the frequency of IL-12 genotype (1188A/C) between different groups (P>0.05).CONCLUSION: The polymorphism of II12B (1188A/C)appears to have some influence on the outcome of HCV infection.
基金Supported by National Natural Science Foundation of China, No 30371618
文摘AIM: To understand the expression of latent and lytic genes of Epstein-Barr virus (EBV) in EBV-associated gastric carcinoma (EBVaGC) and to explore the relationship between EBV-encoded genes and development of EBVaGC at molecular level, METHODS: One hundred and seventy-two gastric carcinoma tissues and 172 corresponding para-carcinoma tissues were tested for EBV genome by polymerase chain reaction (PCR)-Southern blotting. EBV-encoded small RNA (EBER) 1 of the PCR positive specimens was detected by in situ hybridization (ISH). Gastric carcinomas with positive EBER1 signals were classified as EBVaGCs. RT-PCR and Southern hybridization were applied to the detection of expression of nuclear antigen (EBNA) promoters (Qp, Wp and Cp), EBNA 1 and EBNA 2, latent membrane proteins (LMP) 1, 2A and 2B and lytic genes (immediate early genes BZLF1 and BRLF1, early genes BARF1 and BHRF1, late genes BcLF1 and BLLF1) in EBVaGCs. RESULTS: Eleven EBV positive samples existed in gastric carcinoma tissues (6.39%). No EBV positive sample was found in corresponding para-carcinoma tissues. The difference between EBV positivity in carcinoma tissues and corresponding para-carcinoma tissues was significant (x2 = 9.0909, P = 0.0026). Transcripts of Qp and EBNA1 were detected in all the 11 EBVaGCs, while both Wp and Cp were silent. EBNA2, LMP1 and LMP2B mRNA were absent in all the cases, while LMP2A mRNA was detected in 4 of the 11 cases. Of the 11 EBVaGCs, 7 exhibited BcLFl transcripts and 2 exhibited BHRF1 transcripts. The transcripts of BZLF1 and BARF1 were detected in 5 cases, respectively. No BLLF1 and BRLF mRNA were detected. CONCLUSION: The latent pattern of EBV in gastric carcinoma corresponds to the latency I/II. Some lytic infection genes are expressed in EBVaGCs tissues. BARF1 and BHRF1 genes may play an important role in tumorigenesis of gastric carcinoma.
基金Supported by NMRC Grant,No.0415/2000,R-182-000-037-213
文摘AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of Hpyloriwere associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: Hpyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the Hpyloripositive patients, 75% (38/51) had Hpyloriin all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori，irrespective of the clinical diagnosis.
基金Supported by a fellowship (to Zhou B) from Max-Planck-Society, Germany, and partially supported by the National Key Basic ResearchDevelopment Program (973 Program) of China, No. 2002CB513006 (to Zhou B)
文摘AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.
基金the National Natural Science Foundation of China, No.30070020
文摘AIM: To study the effect of co-expression of ppsA, pckA,aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strainfor L-phenylalanineoMETHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coil and Brevibactenurn flavum to generate constructs pJN2 and pJNS.pJN2 was generated by inserting ppsA and pCKA genes into vector pCZ; whereas pJN5 was obtained by introducing ppSA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes.The recombinant plasmids were then introduced into B.flavum by electroporation and the transformants were used for L-phenylalanine fermentation.RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably, pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly.CONCLUSION: Co-expression of ppsA and pckA. genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pcsA, aroG, pheA and tyrB of E. coil in B. flavum was a feasible approach to construct a strain for phenylalanine production.
文摘AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice.METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector,whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid.Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared.RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line.No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01).The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group.CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.
基金supported by research grants from the National Natural Science Foundation of China(No.30271l87)the Shanghai Science and Technology Development Foundation(No.02DJ14015)
文摘The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.
基金Supported by the National High Technology ResearchDevelopment Program of China, No. 2001AA227111
文摘AIM: To characterize the CagA variable region of Helicobacter pylori isolates from Chinese patients.METHODS: DNA fragments in CagA variable region were amplified and sequenced respectively from genomic DNA of 19 isolates from patients with gastric cancer and 20isolates from patients with chronic gastritis. The tendency of phosphorylation in tyrosine(s) of CagA proteins was evaluated subsequently by phosphorylation assay in vivo and in vitro respectively.RESULTS: About 97.44% (38/39) H pylori isolates possessed CagA gene. CagA+ strains contained 2-4tandem five-amino-acid motifs EPIYA but only one EPIYA had repeated sequence in CagA variable region in different isolates. There was no significant difference between the number of EPIYA motifs in H pylori from patients with different diseases. However, only tyrosine site in EPIYA within repeated sequence could be phosphorylated by AGS cells in vivo although all tyrosine sites in EPIYA could be phosphorylated in vitro.CONCLUSION: CagA in Chinese has no functional difference in perturbing cellular signal pathway among different H pylori isolates.
