Recombinant snake venom metalloproteinase inhibitor BJ46A inhibits invasion and metastasis of B16F10 and MHCC97H cells through reductions of matrix metalloproteinases 2 and 9 activities.

Studies have shown that the recombinant BJ46a (rBJ46a) protein can reduce matrix metalloproteinase (MMP) activities and inhibit invasion and metastasis of melanoma cells. Here, we optimized the Pichia pastoris system to evaluate rBJ46a protein as an anticancer agent. The Enzchek gelatinase/collagenase assay showed that rBJ46a inhibited MMP activities (IC50=0.119 mg/ml). Kinetic analyses using a series of double reciprocal Lineweaver-Burk plots (1/V vs. 1/S) showed a competitive mode of inhibition with rBJ46a with inhibitory efficiency against MMPs (Ki=13.6 nmol/l). Matrigel invasion assays showed significant activity of rBJ46a on tumor cells. For lung colonization assays, C57BL/6 mice were inoculated in the lateral tail vein with B16F10 cells and were treated with three i.v. injections of rBJ46a (1, 2, and 4 mg/kg) 24 h before cell inoculation, and 2 and 24 h after cell inoculation. Administration of rBJ46a suppressed lung tumor colony formation significantly. For spontaneous metastasis assays, MHCC97H cells were inoculated subcutaneously into nude mice. After 24 h, rBJ46a was administered by i.p. injections: 1, 2, and 4 mg/kg once daily for 6 days. rBJ46a decreased lung tumor colony formation significantly. Gelatin zymography showed that MMP2/MMP9 enzymatic activities in tumor cells were suppressed by rBJ46a in a dose-dependent manner, and the Km values of rBJ46a against MMP2 and MMP9 activities that were expressed in both B16F10 and MHCC97H cells were 3.6 and 1.4 µmol/l, respectively. Thus, rBJ46a can inhibit the invasion and metastasis of tumor cells by reducing MMP2/MMP9 activities, indicating that rBJ46a may be a novel therapeutic agent for antimetastasis of tumor cells.

PA28ß regulates cell invasion of gastric cancer via modulating the expression of chloride intracellular channel 1.

PA28ß is a subunit of proteasome activator PA28. Previous study suggests that PA28ß is involved in the invasiveness and metastasis of gastric adenocarcinoma (GA), however, the mechanism is not fully understood. In the present study, we showed that invasive abilities of gastric cancer cells were enhanced when PA28ß being down-regulated, and were inhibited when PA28ß being overexpressed. To explore the possible mechanism of PA28ß associated elevated invasiveness, the protein profiles of PA28ß knock down and parental negative control gastric cancer cells were compared using proteomics approach. The results revealed that there were 43 proteins were differentially expressed, among them, chloride intracellular channel 1 (CLIC1) was significantly up-regulated and selected for further functional study. Down-regulation of CLIC1 by RNA interference was able to markedly inhibit cell invasion of PA28ß knock down gastric carcinoma cells. In addition, an inverse correlation between PA28ß and CLIC1 expressions was also verified in GA tissue samples, suggesting that knockdown of PA28ß could enhance tumor invasion and metastasis, at least in part, through up-regulation of CLIC1. Our results provide novel insight into the mechanisms of PA28ß related invasiveness and metastasis of GA, and suggest new alternative approaches for GA treatment.

Overexpression of thrombospondin-1 in stromal myofibroblasts is associated with tumor growth and nodal metastasis in gastric carcinoma.

BACKGROUND AND OBJECTIVES: The roles of thrombospondin-1 (THBS-1) in tumor growth and metastasis are complicated and its function as a cancer inhibitor or promoter remains controversial. This clinical study investigated the functional roles of THBS-1 in gastric carcinoma by examining the expression patterns of THBS-1 protein and mRNA levels during gastric cancer development. METHODS: Eighty-two gastric carcinomas were included in this study. THBS-1, α-smooth muscle actin, and CD34 proteins were localized by immunohistochemical staining, and the levels of THBS-1 mRNA were quantified by real-time polymerase chain reaction. RESULTS: THBS-1 mRNA expression in gastric carcinoma tissues was significantly higher than in adjacent non-cancerous stomach tissues (P = 0.03). Tumor THBS-1 mRNA expression level was significantly related to lymph node metastasis (P = 0.031), tumor size (P = 0.021) and patient age (P = 0.005). THBS-1 protein was mainly located in stromal myofibroblasts, and was undetectable in tumor cells. Myofibroblasts may be mainly derived from stromal fibroblasts in gastric cancer. The abundance of myofibroblasts was positively correlated with tumor growth and nodal metastasis in gastric carcinoma (P = 0.03, P = 0.0008, respectively). CONCLUSIONS: This clinical study revealed that overexpression of THBS-1 in stromal myofibroblasts is associated with tumor growth and nodal metastasis in gastric carcinoma. THBS-1 may activate latent transforming growth factor-ß1 to stimulate fibroblasts to differentiate into myofibroblasts, though further studies are needed to validate this hypothesis. These results suggest that THBS-1 and myofibroblasts may serve as novel targets for strategies aimed at protection against and treatment of gastric carcinoma.

Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice.

A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4 weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50 ± 0.22 to 7.60 ± 0.74 days); induced high levels of IgG antibody (from 0.252 ± 0.080 to 0.790 ± 0.083), IFN-gamma (from 598.74 ± 67.50 to 853.77 ± 66.74 pg/ml), and IL-2 (from 89.44 ± 10.66 to 192.24 ± 19.90 pg/ml); changed the CD4(+)/CD8(+) lymphocyte ratio (from 1.81 ± 0.14 to 1.09 ± 0.19); and stimulated NK cell-killing activity (from 46.81 ± 3.96 to 64.15 ± 7.71 %). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.

Hepatitis B doubly spliced protein, generated by a 2.2 kb doubly spliced hepatitis B virus RNA, is a pleiotropic activator protein mediating its effects via activator protein-1- and CCAAT/enhancer-binding protein-binding sites.

The 2.2 kb doubly spliced defective hepatitis B virus (HBV) genome is frequently detected in the serum of patients with chronic hepatitis B. However, the biological significance of this type of defective genome is not well understood. In this study, expression of the hepatitis B doubly spliced protein (HBDSP) was confirmed from the 2.2 kb doubly spliced defective HBV genome, which was isolated and transfected into Huh-7 hepatoma cells. To explore the potential pathogenicity of HBDSP, hepatocellular proteins interacting with HBDSP were screened by a yeast two-hybrid assay. Unexpectedly, HBDSP could transactivate the GAL4-responsive element, and deletion mapping revealed that the fragment located between residues Leu-48 and Gln-75 of HBDSP was crucial for transactivation activity. In Huh-7 hepatoma cells, HBDSP localized predominantly to the cytoplasm and showed transactivating effects on the cytomegalovirus immediate-early promoter, simian virus 40 enhancer/promoter and HBV regulatory elements including the S1 promoter, S2 promoter, Enhancer I and core upstream regulatory sequences. Further studies revealed that the transactivating activities were mediated by activator protein-1- and CCAAT/enhancer-binding protein-binding sites. These findings suggest that HBDSP is a pleiotropic activator protein that can potentially serve as an HBV virulence factor.

Hepatitis B spliced protein (HBSP) generated by a spliced hepatitis B virus RNA participates in abnormality of fibrin formation and functions by binding to fibrinogen γ chain.

Hepatitis B spliced protein (HBSP) encoded by a 2.2 kb singly spliced hepatitis B virus (HBV) pre-genomic RNA (spliced between positions 2447 and 489 nt) is involved in the pathogenesis of HBV infection, whereas the exact mechanism is far from being fully elucidated. In this study, a yeast two-hybrid system using HBSP as bait was employed to screen binding partners for HBSP from a human liver cDNA library. The interaction between HBSP and fibrinogen γ chain (FGG) was further confirmed in vitro using a GST pull-down assay and confirmed in vivo using a mammalian two-hybrid assay and co-immunoprecipitation. It was identified that this interaction is mediated by the N terminal 47 amino acid residues of HBSP. HBSP could inhibit fibrin polymerization, factor XIIIa-mediated fibrin cross-linking, adhesion of platelets to fibrinogen and ADP-stimulated platelet aggregation. However, the interaction-mediating fragment 1-47 of HBSP is not sufficient for the inhibitory activity on fibrinogen function. The findings suggested that HBSP may participate in the hemostatic abnormality in patients with HBV-related liver diseases.

Toxoplasma gondii: expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages.

In this study, effects of GRA1 organelle-targeted expression on macrophage functions were investigated. The recombinant plasmid pCMV/myc/ER-GRA1 was constructed and then was transfected into murine macrophage RAW264.7 by Lipofectamine, selected by resistance of G418. The selected mono-clone cell line was named ER-GRA1-RAW264.7. The expression of GRA1 was localized in ER of ER-GRA1-RAW264.7 cells by indirect immunofluorescence detection. GRA1 mRNA expression level in ER-GRA1-RAW264.7 cell was significantly enhanced with a concomitant increase in its growth and adherence activity. Fluorescence intensity of intracellular calcium in ER-GRA1-RAW264.7, ER-ctrl-RAW264.7 and RAW264.7 cells in the presence of 1 mmol/l arachidonic acid (AA) were assayed by confocal microscopy using calcium-sensitive dye, Fluo-3 AM. Cytoplasm [Ca2+]i peaked at about 18 s after AA treatment, and cytoplasm [Ca2+]i of RAW264.7 cell almost instantly stepped up after AA was added, and peaked in 3 s, with a minor cytoplasm [Ca2+]i vibration subsequently. These results demonstrated that the expression of GRA1 in ER of macrophages promotes both growth and adherence of macrophages and modulates the intracellular calcium release stimulated by AA.

Identification of miRNAs associated with tumorigenesis of retinoblastoma by miRNA microarray analysis.

INTRODUCTION: MicroRNAs (miRNAs) are small (approximately 22 nucleotides) regulatory RNAs which play fundamental roles in many human diseases, including cancer. There is no report on the miRNA expression profile of retinoblastoma. METHODS: This work was undertaken to identify differentially expressed miRNAs in human retinoblastoma tissues by microRNA microarray technique, and some miRNAs were verified using northern blot analysis and the in situ hybridization method. RESULTS: A cluster of microRNAs was identified as highly expressed in retinoblastoma, including hsa-miR-494, hsa-let-7e, hsa-miR-513-1, hsa-miR-513-2, hsa-miR-518c*, hsa-miR-129-1, hsa-miR-129-2, hsa-miR-198, hsa-miR-492, hsa-miR-498, hsa-miR-320, hsa-miR-503, and hsa-miR-373*. CONCLUSION: These miRNAs are the first to be reported for human retinoblastoma and may play significant roles in regulating tumor genesis.

[A hospital-based case-control study on influencing factors of nonalcoholic fatty liver disease].

OBJECTIVES: To investigate the influencing factors of nonalcoholic fatty liver disease (NAFLD). METHODS: A hospital-based case-control study was conducted in patients with NAFLD and controls without NAFLD in a hospital from January to August in 2007. All data were analyzed by SPSS 13.0 software. RESULTS: One-way analysis of variance found that the two groups were significantly different in cigarette smoking, alcohol and tea comsumption, movement index, speed of food intake, frequency of social engagement, kinds of edible oil, marine products, family history of NAFLD, hypertension, higher blood sugar, abnormality of blood fat, higher level of ALT, higher level of AST, hyperuricemia, obesity, decrease of high density lipoprotein (HDL), and increase of low density lipoprotein. By non-conditional logistic stepwise regression analysis, 12 of 18 factors were used to construct a model, ten of which were the risk factors and two were protective factors of NAFLD. Risk factors included obesity (OR=6.35), hypertension(OR=3.82), dyslipidemia (OR=2.95), decrease of HDL (OR=2.85), hyperglycemia (OR=2.82), increase of ALT (OR=2.80), hyperuricemia (OR=2.35), HBsAg positive (OR=1.99), family history of fatty liver (OR=1.79) and frequently intake of marine products (OR=1.58), and protective factors included tea drinking (OR=0.72) and exercise (OR=0.90). CONCLUSIONS: There are many influencing factors of NAFLD, and life styles are the key factors. Genetic background may also play some roles in NAFLD.

Influence of enterococci on human sperm membrane in vitro.

AIM: To study the influence of enterococci on human sperm membrane in vitro. METHODS: Ejaculated human sperm were artificially infected with beta-hemolytic or non-beta-hemolytic enterococci at the bacteria: sperm ratio of 50:1 at 37 degrees . Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling (HOS) test and electron microscopy. RESULTS: Sperm infected with beta-hemolytic enterococci had lower HOS scores compared with non-beta-hemolytic strains or uninfected control (P < 0.01). The HOS test scores of sperm infected with beta-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-beta-hemolytic strains showed no significant difference in swelling rate, compared with the control group (P > 0.05). It was shown by electron microscopy that beta-hemolytic enterococci caused significant rupture of human sperm membrane. CONCLUSION: Beta-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.

Increased expression of angiogenin in gastric carcinoma in correlation with tumor angiogenesis and proliferation.

AIM: To investigate the implication of angiogenin (ANG) in the neovascularizaton and growth of human gastric carcinoma (HGC). METHODS: ANG mRNA expression in HGC specimens obtained by surgical resection from patients with HGC were examined by RT-PCR. ANG, Ki-67, VEGF protein expression and microvessel density (MVD) in HGC specimens were detected by immunohistochemistry. RESULTS: RT-PCR showed significantly higher ANG mRNA expression (0.482 +/- 0.094) in HGC tissues than in the surrounding nontumorous tissues (0.276 +/- 0.019, P = 0.03). MVD within tumorous tissues increased significantly with ANG mRNA expression (r = 0.380, P = 0.001) and ANG protein expression (P < 0.01). The ANG expression levels of cancer tissues were positively correlated with VEGF (P < 0.01) and the proliferation index of cancer cells (P < 0.01). CONCLUSION: ANG is one of the neovascularization factors of HGC. ANG may work in coordination with VEGF, and promote the proliferation of HGC cells.

[Correlation between the expression of angiopoietins and their receptor and angiogenesis in gastric cancers].

OBJECTIVE: To explore the effects of angiopoietins (Ang-1 and Ang-2) and Tie-2 expression on microvessel density (MVD) in gastric cancers. METHODS: By using semiquantitative RT-PCR, immunohistochemistry and image analysis system, the expression of Ang-1, Ang-2, Tie-2 mRNA and their proteins were detected in 68 primary gastric cancers and their adjacent normal tissues. Microvessel density (MVD) was figured out based on CD34 immunohistochemical staining. RESULTS: The expression of all Ang-1, Ang -2, Tie-2 mRNA and their proteins was detected in gastric cancers and their paired adjacent gastric mucosa tissues. A negative correlation between Ang-1 protein, Tie-2 mRNA and MVD in gastric cancers was observed (r = -0.440, r = -0.267; P < 0.05), while the relation between Ang-2 mRNA and its protein, Ang-2/Ang-1 protein ratio with MVD were positive (r = 0.319, r = 0.729, r = 0.739; P < 0.05). It was found that MVD in groups with Ang-2 mRNA T/N ratio over 1.2 (the ratio of Ang-2 mRNA in gastric cancers and its adjacent normal mucosa) was higher than that in those with a ratio under 1.2, revealed by analysing the effects of Ang-1 and Ang-2 mRNA T/N ratio on MVD in gastric cancers. CONCLUSION: Ang-1 activates Tie-2 receptor, whereas Ang-2 antagonizes Ang-1 in the angiogenesis, and the Ang-2/Ang-1 ratio determines angiogenesis and tumor growth in gastric cancers. When the expression of Ang-2 is high and Ang-1 is low, the angiogenesis in gastric cancers is promoted, otherwise oppositely. The role of Ang-2 is dominant in the effect of Angs and their receptor on angiogenesis in gastric cancers.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated.

AIM: To investigate the biological impacts of "hot-spot" mutations on genotype B and C HBV X proteins (HBx). METHODS: Five types of "hot-spot" mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I, I127T+K130M+V131I, or K130M+V131I+F132Y, respectively, were generated by means of site-directed mutagenesis. To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity, and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVbeta to Chang cells, which were lysed 48 h post-transfection and the intra-cellular beta-galactosidase activities were monitored (increase of the beta-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test. RESULTS: As compared to standard genotype B HBx, mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity, compared to standard genotype C HBx, F132Y and K130M+ V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity, while the mutants of I127T and I127T+K130M+V131I showed no differences. CONCLUSION: "Hot-spot" mutations affect the anti-proliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most "hot-spot" mutations on HBx are genotype B and C differentiated.

Association of THBS1 rs1478605 T>C in 5'-untranslated regions with the development and progression of gastric cancer.

Thrombospondin 1 (THBS1) plays an important role in angiogenesis and tumor progression. The aim of the present study was to investigate the effects of single-nucleotide polymorphisms (rs1478605 and rs3743125) in the untranslated regions of the THBS1 gene on the development and progression of gastric cancer. In the case-control study, 275 gastric cancer patients and 275 cancer-free controls were successfully genotyped using polymerase chain reaction-restriction fragment length polymorphism. The data demonstrated that THBS1 rs1478605 genotypic distributions significantly differed between the patient and control groups (P=0.005). Carriers of the CC genotype exhibited a decreased risk of developing gastric cancer compared to the carriers of the CT and TT genotypes [adjusted odd ratio (OR), 0.56; 95% confidence interval (CI), 0.39-0.79; P=0.001]. The CC genotype of rs1478605 was negatively associated with gastric cancer lymph node metastasis (OR, 0.41; 95% CI, 0.23-0.71; P=0.001) and was associated with a reduced risk of lymph node metastasis in male patients (OR, 0.27; 95% CI, 0.14-0.52; P<0.001). The THBS1 CT haplotype was associated with a reduced risk of developing gastric cancer (OR, 0.56; 95% CI, 0.33-0.93; P=0.02). By contrast, no association was observed between THBS1 rs3743125 and the development and progression of gastric cancer. These results suggest that THBS1 rs1478605 represents a potential molecular marker for gastric cancer.

Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma.

AIM: The expressive balance between matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor of metalloproteinase-1 (TIMP-1) plays a critical role in maintaining the degradation and synthesis of extracellular matrix. Loss of such balance is associated with invasion and metastasis of tumors. This study aimed to determine the expression of MMP-9 and TIMP-1 in gastric carcinoma, and the association of the expressive imbalance between MMP-9 and TIMP-1 with the invasion and metastasis and prognosis of gastric carcinoma. METHODS: We used immunohistochemistry to determine the expressions of MMP-9, TIMP-1 and proliferating cell nuclear antigen Ki-67 in the gastric specimens taken from 256 patients with primary gastric carcinoma. The patients were followed-up for up to 96 months. RESULTS: No association between the expression of MMP-9 and TIMP-1 and patients' sex and age, tumor size and location of gastric carcinoma was observed. The incidence of the positive expression of MMP-9 in cases with tumors invasion to muscularis propria and visceral peritoneum (70.13 % and 69.09 %, respectively) was significantly higher than that in cases with tumor invasion only to lamina propria or submucosa (42.50 %, P=0.0162). The positive correlation between MMP-9 expression and the depth of tumor invasion was observed (Pearson correlation coefficient=0.2129, P=0.016). Along with the increase of the metastatic station of lymph nodes, the incidence of the MMP-9 expression was increased by degrees; a positive correlation between them was observed (Pearson correlation coefficient=0.2910, P=0.0001). There was also a significant correlation between MMP-9 expression and the TNM stage in gastric carcinoma (Pearson correlation coefficient=0.3027, P<0.0001). The incidence of MMP-9 expression in stage II and III/IV (75.00 % and 76.15 %, respectively) was significantly higher than those in stage I (46.15 %, P<0.0001). A negative correlation between TIMP-1 immunoreactivity and the depth of invasion, status of lymph node metastasis and TNM stage was observed (Pearson correlation coefficient =-0.1688, -0.3556 and -0.3004, P=0.023, <0.0001 and <0.0001, respectively). Four types of co-expression of MMP-9 and TIMP-1 were observed; i.e. MMP-9 positive but TIMP-1 negative (n=115), both positive (n=52), both negative (n=62) and MMP-9 negative but TIMP-1 positive (n=27). The frequency of serosal invasiveness was significant higher in patients with MMP-9 but without TIMP-1 expression than those with other types of the co-expression (P=0.0303). The incidence of lymph node metastasis was highest in patients with MMP-9 but without TIMP-1 expression, and lowest in those with TIMP-1 but without MMP-9 expression (P<0.0001). The survival rate in patients with MMP-9 but without TIMP-1 expression was lower than that in those with TIMP-1 but without MMP-9 expression (P=0.0014). CONCLUSION: Our results in gastric carcinoma demonstrated a significant positive association of MMP-9 over-expression with proliferation of tumor cells, the depth of invasiveness, lymph node metastasis and TNM stage, suggesting MMP-9 can serve as a molecular marker of tumor invasion and metastasis. We also demonstrate a significant negative relationship of TIMP-1 expression with the depth of invasiveness and lymph node metastasis, which provide a new idea in the tumor biological and genetic treatment. The interaction between MMP-9 and TIMP-1 in the processes of tumor invasion and metastasis is that MMP-9 mainly promotes tumor invasion and metastasis and TIMP-1 inhibits functions of MMP-9. The imbalance between MMP-9 and TIMP-1 expression may suggest the occurrence of tumor invasion and metastasis, predict poor prognosis. For patients with imbalanced MMP-9 and TIMP-1 expression, the optimal treatment scheme needs to be selected.

Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice.

AIM: To explore the virulence and the infectivity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to sterile tap water. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid form by exposure to sterile tap water. Both spiral and coccoid forms of H. pylori were tested for the urease activity, and the adherence to Hep-2 cells. The presence of flagella was examined under electron microscopy. In the experimental animal infection, the spiral and coccoid forms of H. pylori originated from the same strain F49 were inoculated intragastrically into BALB/c mice respectively four times at a 3-day interval. Half of the mice from each group were sacrificed at Day 21 and Day 28 after the last inoculation. Histology and H. pylori colonization were detected by urease test of gastric mucosa, cultures of H. pylori, and electron microscopy and so on. RESULTS: The urease activity and the ability of adherence to Hep-2 cells were found to be lower in coccoid H. pylori than that in its spiral form. For example, the transformation in strain F(44) led to a significant decrease of the adherence rate and adherence index from 70.0+/-5.3 % to 30.2+/-3.5 % (P<0.01), and from 2.6+/-0.4 to 0.86+/-0.3 (P<0.01), respectively. The flagella of coccoid H. pylori were observed under electron microscope. In the experimental infection in mice, the positive rate of gastric mucosa urease test was 93.8 % (15/16) in the group infected by spiral H. pylori and 50 % (8/16) in the group infected by coccoid H. pylori, and the estimated coccoid H. pylori colony number was 1.75 vs 0.56. The positive rates of H. pylori culture were 87.5 % (14/16) in spiral H. pylori group and 68.8 % (11/16) in coccoid H. pylori group. There was no significant difference in either urease test or bacterial culture rate between the groups examined at Day 21 and Day 28 after inoculation. Electron microscopic examination of the samples taken from both groups showed the adherence of H. pylori in spiral, bacillary and coccoid shapes to the epithelial cells of gastric wall. Histological examination showed the occurrence of gastric mucosal injury as indicated by various degrees of erosion, ulcer, and inflammatory cell infiltration. Mucosal injury was slighter in the mice infected by coccoid H. pylori. No positive result was obtained in the control group that received intragastrical administration of sterile tap water. CONCLUSION: Although the virulence of coccoid H. pylori induced by water decrease, coccoid H. pylori still remains a considerable urease activity and the adhering ability to epithelial cells. Furthermore, the flagella, an important component responsible for bacterial movement and infection, were still observed as a cellular structure of coccoid H. pylori under electron microscope. The coccoid H. pylori induced by water is capable of colonizing in gastric mucosa and causing gastrititis in mice.

Seek protein which can interact with hepatitis B virus X protein from human liver cDNA library by yeast two-hybrid system.

AIM: To seek the X associated protein (XAP) with the constructed bait vector pAS2-1X from normal human liver cDNA library. METHODS: The X region of the HBV gene was amplied by PCR and cloned into the eukaryotic expression vector pAS2-1. The reconstituted plasmid pAS2-1X was transformed into the yeast cells and the expression of X protein (pX) was confirmed by Western blot analysis. Yeast cells were cotransformed with pAS2-1X and the normal human liver cDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with the LacZ report gene. Furthermore, segregation analysis and mating experiment were performed to eliminate the false positive and the true positive clones were selected for PCR and sequencing. RESULTS: Reconstituted plasmid pAS2-1X including the anticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed that reconstituted plasmid pAS2-1X expressed BD:X fusion protein in yeast cells. Of 5 x 10(6) transformed colonies screened,65 grew in the selective SC/-trp-leu-his-ade medium, 5 scored positive for beta-gal activity, and only 2 remaining clones passed through the segregation analysis and mating experiment. Sequence analysis identified that two clones contained similar cDNA fragment:GAACTTGCG. CONCLUSION: The short peptide(glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normal human liver cDNA library has difficulties in expressing the integrated XAP on yeast cells.

[Dual effect of angiopoietin-2 on angiogenesis in gastric cancer].

OBJECTIVE: To explore the effect of Ang-2 on angiogenesis in gastric cancer. METHODS: The expression of Ang-2 mRNA was analyzed by RT-PCR and the expression of VEGF and CD34 was detected by immunohistochemistry in 36 cases of gastric cancer tissues and their paired adjacent gastric mucosa. RESULTS: The expression of Ang-2 mRNA was found both in gastric cancer and their paired adjacent gastric mucosa; the correlationship between the general expression levels of Ang-2 mRNA and microvessel density (MVD) in gastric cancer tissues was not found. However, in 27 cases whose Ang-2 mRNA expression levels in cancer tissues were lower than those in adjacent gastric mucosa. A significant positive correlation between the expression level of Ang-2 mRNA and MVD in the tumor tissues was found (r = 0.411, P < 0.05). In these 27 cases, the MVD in the gastric cancer tissues with positive VEGF expression (45.45 +/- 10.30) was higher than that with negative VEGF expression (30.15 +/- 8.69, P < 0.05), whereas in the other 9 cases whose expression levels of Ang-2 mRNA in cancer tissues were higher than those in adjacent gastric mucosa, a significant negative correlation between expression level of Ang-2 mRNA and MVD in the tumor tissues (r = -0.758, P < 0.05), but without correlation between the MVD and VEGF. CONCLUSION: Under the conditions that the expression of Ang-2 mRNA in cancer tissues was lower than that in adjacent gastric mucosa, VEGF could promote the sprouting of new vessels along with Ang-2 upregulation. But under the conditions that the expression of Ang-2 mRNA in cancer tissues was higher than that in adjacent gastric mucosa, Ang-2 inhibited angiogenesis. Angiopoietin-2 may play a dual effect on angiogenesis in gastric cancer.

[Expression and significance of nitric oxide synthase in human hepatocellular carcinoma].

OBJECTIVE: To investigate the expression and significance of nitric oxide synthase (cNOS) mRNA in primary hepatocellular carcinoma (HCC), cirrhotic liver and normal liver tissue. METHODS: cNOS mRNA expression in 80 HCC, 40 cirrhotic liver and 20 normal liver tissue were observed by in situ hybridization. CD34 immunostain was used to measure the microvascular density (MVD) and Ki-67 immunostain to proliferative index. RESULTS: Expression of cNOS mRNA was observed in the liver cancer cells, endothelial cells in the non-cancerous liver tissues and mononuclear and/or phagocytes. Expression of cNOS mRNA in tumor cells of HCC was higher than that in the liver cells of cirrhotic liver (P < 0.01) which was higher than the normal liver tissue. Expression in the endothelial cells was higher in HCC and cirrhotic liver than those in the normal liver tissue (P < 0.01). HCC with positive cNOS mRNA expression in the endothelial cells showed higher extent of neovascularization and degree of proliferative index. The more MVD, the higher proliferative index, which increased in metastatic tumors. CONCLUSION: cNOS mRNA expression was involved in oncogenesis, angiogenesis and progression of hepatocellular carcinoma.

[Structural analysis of 22 full-length hepatitis B virus genomes isolated from patients with hepatocellular carcinoma].

OBJECTIVE: To study the characteristics of full-length Hepatitis B Virus (HBV) genomes isolated from patients with hepatocellular carcinoma (HCC). METHODS: The full-length HBV genomes from the serum of HBsAg positive HCC patients were amplified by PCR, and then sequenced and analyzed its structure. RESULTS: Twenty-two full-length HBV DNAs were obtained from different patients of HCC. Phylogenetic analysis revealed that all HBV strains could be categorized into genotype B or C and serotype adr or adw2. Structural analysis showed that HBV obtained shared meaningful consensus mutations in B/T cell epitopes of surface and core antigens, transactivating domain of X protein and enhancer II/core promoter regions as compared to standard strains. CONCLUSION: Genotype and gene mutation of HBV may be closely correlated with the carcinogenesis of HBV-related hepatocellular carcinoma.