Copper-catalyzed, stereoconvergent, cis-diastereoselective borylative cyclization of ω-mesylate-α,ß-unsaturated esters and ketones.

The Cu(i)-catalyzed stereoconvergent borylative cyclization of ω-mesylate-α,ß-unsaturated compounds is facilitated by a simple Cu-bisphosphine catalyst. This reaction provides a novel route to cis-ß-boron-substituted five- and six-membered carbocycle and heterocycle esters. Mechanistic studies indicate that stereoconvergence and cis-substitution likely stem from the rapid enolation of the borylcopper adduct with the substrate double bond and the formation of a five-membered intermediate, respectively.

Effect of 1,25-(OH)2D3 on Proliferation of Fibroblast-Like Synoviocytes and Expressions of Pro-Inflammatory Cytokines through Regulating MicroRNA-22 in a Rat Model of Rheumatoid Arthritis.

OBJECTIVE: This study aims to investigate the regulatory mechanism of 1,25-(OH)2D3 on the proliferation of fibroblast-like synoviocytes (FLS) and expressions of pro-inflammatory cytokines in rheumatoid arthritis (RA) rats via microRNA-22 (miR-22). METHODS: A rat model of RA was established with a subcutaneous injection of type II collagen. After treated with different concentrations of 1,25-(OH)2D3 the proliferation of FLS was estimated by the MTT method, and the optimal concentration of 1,25-(OH)2D3 was selected for further experiments. Cell proliferation was detected by MTT. Cell cycle and apoptosis were analyzed by FCM. The IL-1ß, IL-6, IL-8, and PGE2 protein expressions were determined by ELISA, and MMP-3, INOS, and Cox-2 mRNA expressions were measured by qRT-PCR. RESULTS: The rat model of RA was successfully established. Compared with the blank group, the 1,25-(OH)2D3 and miR-22 inhibitors groups exhibited higher proliferation inhibition and apoptosis rates, lower levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and PGE2), and decreased mRNA expressions of MMP-3, INOS, and Cox-2. The miR-22 mimics group had lower proliferation inhibition and apoptosis rates, elevated expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 than the blank group. In contrast to the 1,25-(OH)2D3 group, the proliferation inhibition and apoptosis rates were down-regulated, and the expressions of pro-inflammatory cytokines and MMP-3, INOS, and Cox-2 were up-regulated in the 1,25-(OH)2D3 + miR-22 mimics group. CONCLUSION: Our study demonstrated that 1,25-(OH)2D3 inhibits the proliferation of FLS and alleviates inflammatory response in RA rats by down-regulating miR-22.

Recombinant adeno-associated virus carrying thymosin ß4 suppresses experimental colitis in mice.

AIM: To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin ß4 (AAV-Tß4) on murine colitis via intracolonic administration. METHODS: AAV-Tß4 was prepared and intracolonically used to mediate the secretory expression of Tß4 in mouse colons. Dextran sulfate sodium (DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to establish a mouse colitis model resembling Crohn's disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tß4 on colitis. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis and proliferation were detected by TUNEL assay and immunochemistry, respectively. RESULTS: Recombinant AAVs efficiently delivered LacZ and Tß4 into the colonic tissues of the mice, and AAV-Tß4 led to a strong expression of Tß4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tß4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tß4 significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tß4 also modulated colonic TNF-α, IL-1ß and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice. CONCLUSION: Tß4 exerts a protective effect on murine colitis, indicating that AAV-Tß4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.

A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice.

Connective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138-159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1-149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased α-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-ß1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.

Prevalence and possible risk factors of low bone mineral density in untreated female patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation. Different studies have shown decreased bone mineral density (BMD) in patients with SLE. The objective of this study was to investigate the prevalence and possible risk factors of low BMD in untreated female patients with SLE in Chinese population. A total of 119 untreated female patients with SLE were included. BMD was measured at lumbar spine and at total hip by dual-energy X-ray absorptiometry. The associations between decreased BMD and demographic variables, clinical variables, and bone metabolism variables were analyzed. These SLE patients had the following characteristics: mean age was 32.6 ± 11.9 years, mean disease duration was 22.1 ± 34.5 months, and mean SLEDAI was 11.4 ± 5.4. Osteopenia was present in 31.1% of the patients and osteoporosis in 8.5%. A significant negative association between low density lipoprotein cholesterol (LDL-c) and BMD at the lumbar spine (correlation coefficient = -0.242; P = 0.023) and total hip (correlation coefficient = -0.259; P = 0.019) was shown. These results seem to indicate that increased LDL-c may be an important risk factor for low BMD at lumbar spine and total hip in untreated female SLE patients.

Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.

Oral administration of recombinant adeno-associated virus-mediated bone morphogenetic protein-7 suppresses CCl(4)-induced hepatic fibrosis in mice.

Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Transforming growth factor-ß1 (TGF-ß1) is the key profibrotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-ß1 and an antifibrotic factor. In this study, we generated a recombinant adeno-associated virus carrying BMP-7 (AAV-BMP-7) and tested its ability to suppress carbon tetrachloride (CCl(4))-induced hepatic fibrosis when orally administered to mice. Our results show that the ectopic expression of BMP-7 in gastrointestinal (GI) mucosa due to the AAV-BMP-7 administration led to the long-term elevation of serum BMP-7 concentrations and resulted in the drastic amelioration of CCl(4)-induced hepatic fibrosis in BALB/c mice. Immunostaining for α-smooth muscle actin (α-SMA) and desmin demonstrated that AAV-BMP-7 inhibited the activation of hepatic stellate cells (HSCs) in the fibrotic mouse liver. Moreover, the ectopic expression of BMP-7 promoted hepatocyte proliferation, as confirmed by an increase in the amount of proliferating cell nuclear antigen (PCNA)-positive hepatocytes in the mice that received AAV-BMP-7. Our results clearly indicate that BMP-7 is capable of inhibiting hepatic fibrosis and promoting hepatocyte regeneration. We suggest that oral AAV-BMP-7 could be developed into a safe, simple, and effective therapy for hepatic fibrosis.

Vaccination with platelet-derived growth factor B kinoids inhibits CCl4-induced hepatic fibrosis in mice.

Platelet-derived growth factor B (PDGF-B) plays an essential role in hepatic fibrosis. Inhibition of the PDGF-B signaling in chronically injured livers might represent a potential therapeutic measure for hepatic fibrosis. In this study, we assessed the effects of vaccination against PDGF-B on CCl4-induced liver fibrosis in BALB/c mice. The PDGF-B kinoid immunogens were prepared by cross-linking two PDGF-B-derived B-cell epitope peptides [PDGF-B¹6-(23-38) and PDGF-B¹6-(72-83)] to ovalbumin and keyhole limpet hemocyanin, respectively. Enzyme-linked immunosorbent assay, Western blotting, and NIH3T3 cell proliferation assay verified that immunization with the PDGF-B kinoids elicited the production of high levels of neutralizing anti-PDGF-B autoantibodies. The vaccination markedly alleviated CCl4-induced hepatic fibrosis, as indicated by the lessened morphological alternations and reduced hydroxyproline contents in the mouse livers. Moreover, immunohistochemical staining for proliferating cell nuclear antigen, α-smooth muscle actin, and desmin demonstrated that neutralization of PDGF-B inhibited both the proliferation and the activation of hepatic stellate cells in the fibrotic mouse livers. Taken together, this study demonstrated that vaccination with PDGF-B kinoids significantly suppressed CCl4-induced hepatic fibrosis in mice. Our results suggest that vaccination against PDGF-B might be developed into an effective, convenient, and safe therapeutic measure for the treatment of hepatic fibrosis.

Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures.

BACKGROUND: The transplantation of neural stem cells (NSCs) has been accepted as a promising therapeutic strategy for central nervous system disorders. However, the beneficial effect of NSC transplantation upon functional recovery is limited due to the unfavorable microenvironment (niche) at the site of trauma or degenerative disease in the brain. Combination of transplantation of NSCs with neurotrophins may overcome the hurdles of impaired cell survival and neuronal differentiation. MATERIAL/METHODS: In the current study, the neurotrophin-3 (NT-3) gene was transduced into cultured mouse embryonic cortical NSCs via an AAV vector (NSC-NT-3). The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.
RESULTS: The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved. Cells in the NSC-NT-3 group proliferated faster and differentiated into more ß-tubulin III-positive neurons compared to the control group in vitro. Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures. CONCLUSIONS: Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs. This work also offers evidence to better understand the safety and efficiency of combined treatment with NT-3 and NSCs for the central nervous system disorders.

Screening of specific binding peptide targeting blood vessel of human esophageal cancer in vivo in mice.

BACKGROUND: Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer. METHODS: The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and VIII-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test. RESULTS: The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P < 0.01 in tumor and spleen, P < 0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or VIII-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker factor VIII-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1.0 × 10(11) pfu/ml was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues (lung and spleen). CONCLUSION: Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer.

Coal cinder filtration as pretreatment with biological processes to treat pharmaceutical wastewater.

This study aims at coupling coal cinder filter with biological process to improve pharmaceutical wastewater quality and reduce the disposal cost. In the coal cinder filter, the removal efficiencies of COD, BOD(5), SS and color were 90+/-2%, 72+/-2%, 95+/-2% and 80+/-2%, respectively. The results attribute to the big specific surface area and strong adsorption ability. Coal cinder filter removes a large portion of the pollutants in the influent wastewater, which would strongly stable the effluent waste water quality, and reduce the load of follow-up biological treatment process. The average removal efficiencies for COD, BOD(5), SS and color of the combined process were about 99.7+/-3%, 98.2+/-4%, 98.5+/-3% and 96.3+/-2%, respectively, with the average effluent quality of COD 16+/-1 mg/L, BOD(5) 11+/-1 mg/L, SS 10+/-0.6 mg/L and color 22+/-1 (multiple), which are consistent with the national requirements of the waste pollutants for pharmaceutical industry of chinese traditional medicine discharge standard (GB 21906-2008). The results indicated that the combined procedure could offer an attractive solution for pharmaceutical wastewater treatment with considerable low cost.

Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats.

AIM: To investigate the potential protective effect of HO-1 on cirrhotic liver cells in rats. METHODS: Male Wistar rats included in the current study were randomly divided into 5 groups as follows: normal (N) group; liver cirrhotic (LC) group; sham (S) group; I/R group and I/R + hemin group. The model for inducing liver cirrhosis in rats was established according to a previously published protocol. Following this the segmental hepatic ischemia reperfusion operation was carried out. The rats were treated with 30 micromol/kg hemin (HO-1 inducer, ferric portoporphyrin IX chloride) i.p. or 0.9% NaCl (control) 24 h and 12 h before hepatic ischemia for 30 min or sham laparotomy. Blood was collected for serum enzymatic measurement 6 and 12 h after reperfusion or sham laparotomy. HO-1, NF-kappaB and caspase-3 expressions were assessed by immunohistochemical analysis. RESULTS: The expressions of proteins are inversely correlated to the gray values. HO-1 expression in the I/R + hemin group was increased significantly than I/R group at 6 h and 12 h after hepatic I/R (6 h: 112.0 +/- 8.3 vs 125.1 +/- 5.7, P < 0.01; 12 h: 120.8 +/- 11.0 vs 132.4 +/- 6.2, P < 0.01). Hemin improved serum manganese superoxide dismutase (MnSOD) (6 h: 131.3 +/- 17.6 vs 107.0 +/- 13.9, P < 0.01; 12 h: 141.4 +/- 12.5 vs 118.3 +/- 10.2, P < 0.01), lessened liver cell injury, decreased caspase-3(6 h: 166.7 +/- 8.1 vs 145.5 +/- 14.6, P < 0.01; 12 h: 172.8 +/- 3.8 vs 148.0 +/- 6.5, P < 0.01) and NF-kappaB expression (6 h: 150.2 +/- 8.6 vs 139.7 +/- 6.0, P < 0.01; 12 h: 151.1 +/- 5.9 vs 148.1 +/- 5.3, P > 0.05) and serum alanine aminotransferase (ALT) (6 h: 413.3 +/- 104.1 vs 626.8 +/- 208.2, P < 0.01; 12 h: 322.2 +/- 98.8 vs 425.8 +/- 115.4, P < 0.05), aspartate aminotransferase (AST) (6 h: 665.2 +/- 70.1 vs 864.3 +/- 70.4, P < 0.01; 12 h: 531.1 +/- 98.6 vs 664.4 +/- 115.6, P < 0.01), malondialdehyde (MDA) levels (6 h: 11.1 +/- 2.17 vs 13.5 +/- 2.01, P < 0.01; 12 h: 9.36 +/- 1.10 vs 10.8 +/- 1.62, P < 0.05) in the I/R + hemin group when compared with the I/R group. CONCLUSION: These results suggest that HO-1 plays an important role in protecting liver cells from hepatic I/R injury in cirrhotic rats by decreasing oxidative stress, apoptosis and inflammation.

[A searching system of parasite pictures and relevant information].

Over 3,000 pictures on five major parasites (schistosome, filaria, hookworm, leishmania and plasmodium) collected between 1950 and 1990 were edited and a searching system was established. The data can be used with a network-based version through a LAN system in the Institute. The adoption of Digital Computerized Management makes it possible for sharing resources in human parasitology.

[Detecting the activity of antibodies induced by recombinant TGFbeta1 vaccine].

OBJECTIVE: To measure the neutralization activity in vitro of the antibodies induced by recombinant TGFbeta1 vaccine and to evaluate the vaccine's anti-liver fibrosis activity. METHODS: Balb/c mice were immunized with a fusion protein of the human TGFbeta1 epitope-inserted into a hepatitis B core antigen using a prokaryotic expression system. The antibody produced by the recombinant vaccine was determined using ELISA. The biological activity of the anti-TGFbeta1 antibody induced by the vaccine was measured by MTT using mink lung epithelial cell Mv-1-Lu as inhibiting cells. The fusion protein was used as a vaccine in a mice hepatic-fibrosis model. RESULTS: A high titer of anti-TGFbeta1 antibody and a low of anti-HBc antibody were detected in the mice after the immunization. The serum antibodies induced combined with the fusion and antigenic peptide prevented the TGFbeta1 inhibiting activity in the Mv-1-Lu cell. CONCLUSION: Recombinant fusion protein can be used as a cytokine vaccine to induce high titers of anti-TGFbeta1 antibodies. Our results show the potentiality of the fusion protein to be used as a vaccine for preventing liver fibrosis.

Intensive inhibition of hTERT expression by a ribozyme induces rapid apoptosis of cancer cells through a telomere length-independent pathway.

Restoration of telomerase activity is essential for most of the malignancies. Telomerase reverse transcriptase (TERT) is the key component of telomerase. In this study, we designed a hammerhead ribozyme against human telomerase reverse transcriptase (hTERT) and observed its growth inhibition and pro-apoptosis effects on cancer cells. The efficiency of this ribozyme was verified in in vitro cleavage experiment. A recombinant retrovirus was constructed to transduce the ribozyme to telomerase positive colon carcinoma cell line SW480 and gastric carcinoma cell line SGC7901. We found that the ribozyme could strongly inhibit hTERT expression and telomerase activity, resulting in rapid apoptosis of cancer cells. Shortening of telomere and replicative senescence were not observed before cell death, indicating intensive inhibition of hTERT expression can induce apoptosis by some mechanism(s) except telomere shortening and replicative senescence. This study suggests that hTERT exerts a direct antiapoptotic function in cancer cells. Anti-hTERT ribozyme might be a potential means in the therapy of telomerase-positive malignancies.

Correlation between a gene polymorphism of tumor necrosis factor and inflammatory bowel disease.

OBJECTIVES: To analyze polymorphism of the tumor necrosis factor (TNF) gene in inflammatory bowel disease (IBD) patients from the Han Chinese ethnic group, and to investigate the role of polymorphism in the pathogenesis of IBD. METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques were used to analyze gene polymorphisms in the TNF-alpha and TNF-beta genes in 131 patients with IBD. RESULTS: The genotype frequency and allelic frequency of TNF-alpha-308 in patients with ulcerative colitis (UC) were 15.5% and 8.7%, respectively, significantly higher than the control population (4.1% and 2.0%, respectively; P < 0.001). There was no significant difference between patients with Crohn's disease (CD) and the normal population with regard to the genotype frequency and allelic frequency of TNF-alpha-308, and neither were there any differences with regard to TNF-beta+252 in patients with IBD (UC and CD) and the normal population. The TNF-alpha-308 polymorphism and the TNF-beta+252 loci did not correlate with age, gender, disease activity or lesion site for IBD patients. CONCLUSIONS: The TNF-alpha-308 allele may be related to susceptibility to UC. The TNF-alpha-308 gene polymorphism is not involved in pathogenesis of CD. No correlation was found between the TNF-beta+252 polymorphism and IBD. Polymorphisms of the TNF-alpha-308 and TNF-beta+252 loci do not correlate with age, gender, disease activity or lesion site.

[Fusion of TGF-beta 1 antigenic determinant and HBV core antigen as an anti-TGF-beta 1 vaccine].

OBJECTIVES: To examine the expression and purification of the TGFbeta1 vaccine from prokaryotic expression system and to determine the antigenicity of the fusion protein of recombinant vector pET28a/ HBcAg1-71-TGFbeta132-HBcAg89-144. METHODS: The reconstructed vector pGEMEX-1/CTC was subcloned to pET28a and transformed into E. coli BL21 (DE3). The recombinant 6xHis- HBcAg1-71- TGFbeta132- HBcAg89-144 was to be expressed after induction by IPTG and purified with Ni-NTA-His affinity chromatography. The detection of the formation of core-like particles was done under an electron microscope and of their antigenity by using ELISA and Western blot. RESULTS: A 2.46 x 10(4) protein was obtained by optimizing the conditions for both expression and purification. The protein had the TGFbeta1 antigenicity but not a HBc antigenity and the formed core-like particles were bigger than natural core particles. CONCLUSION: The recombinant fusion protein in the prokaryotic expressed system can be used as an anti-TGFbeta1 vaccine to inhibit hepatic fibrosis.

[Osteopontin expression and its relation to invasion and metastases in gastric cancer].

OBJECTIVE: To investigate the correlation between expression of the osteopontin (OPN) and invasion and metastases in gastric cancer. METHODS: The expression of OPN, NF-kappaB p65 and matrix metallo-proteinase 9 (MMP-9) was detected by immunohistochemistry in non-cancer gastric tissue (n = 12 cases) and gastric cancer tissue (n = 72 cases). RESULTS: (1) OPN, NF-kappaB p65 and MMP-9 were not expressed in 12 non-cancer gastric tissue samples(group A). Their expression rates were 43.3%, 40.0% and 46.7% respectively in 30 gastric cancer samples without lymph nodes metastasis (group B), but they increased to 76.9%, 73.1% and 80.8% in 26 gastric cancer samples with lymph nodes metastases (group C), and 87.5%, 81.3% and 93.8% respectively in 16 gastric cancer samples with lymph node and distant metastases (group D). (2) There were statistically significant differences in their expressions between group D and group B (P(a) = 0.004, P(c) = 0.007, P(e) = 0.002), and between group C and group B (P(b) = 0.011, P(d) = 0.013, P(f) = 0.009). (3) Despite some differences in positive expression rates, correlations existed between OPN and NF-kappaB p65, and between NF-kappaB p65 and MMP-9 (P(1) = 0.042, P(2) = 0.013; r(1)= 0.67, r(2)= 0.72). CONCLUSION: Osteopondin espression is closely related to the invasion and metastases of gastric cancer. It may upregulate the expression of metastasis-related molecule MMP-9 by activating NF-kappaB pathway.

The potent inhibitory effects of cisapride, a specific blocker for human ether-a-go-go-related gene (HERG) channel, on gastric cancer cells.

BACKGROUND: Ion channels may play a role in carcinogenesis. Human ether-a-go-go-related gene (HERG) encoding one of the components of delayed rectifier potassium currents has been indicated to be involved in tumor cell growth and death. Our aim is to investigate the effects of cisapride, a specific blocker for HERG channel, on human gastric cancer cells. METHODS: The effects of cisapride on the proliferation, clonogenicity, cell cycle and apoptosis of gastric cancer cells were evaluated by MTT assay, clonogenicity assay, flow cytometry and transmission electron microscopy. The expression of HERG mRNA and protein in gastric cancer cells and tissues was measured by RT-PCR, Western blot and immunohistochemistry, respectively. RESULTS: HERG mRNA and protein were exclusively expressed in gastric cancer cells. The HERG protein was localized in the cytoplasm and membrane of the gastric cancer cells. The proliferation of gastric cancer cells expressing HERG protein was inhibited in a time- and dose-dependent manner when treated with cisapride (P<0.05). The clonogenicity of gastric cancer cells treated with cisapride (100 nM) was reduced (P<0.05). Flow cytometric analysis indicated that cisapride tends to inhibit gastric cancer cells entering S phase from G(1) phase in the cell cycle (P<0.05). Apoptotic cells were found increased in gastric cancer cells treated with cisapride by both flow cytometry and electron microscopy. CONCLUSIONS: As HERG channel blocker, cisapride, can inhibit the growth of gastric cancer cells by altering distribution of cell cycle and inducing apoptosis so as to be of potential value in the treatment of gastric cancer.

Construction of prokaryotic expression system of TGF-beta1 epitope gene and identification of recombinant fusion protein immunity.

AIM: To insert the constructed TGF-beta(1) epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-beta(1) antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti-TGF-beta(1) vaccine. METHODS: The TGF-beta(1) encoding epitope gene (the mature TGF-beta(1) from 78-109 amino acid residues, TGF-beta(1)(32)) was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-beta(1)(32) vector. The HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-beta(1)(32) and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T(4) ligase. The fusion gene fragments HBcAg1-71-TGF-beta(1)(32)- HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification with Ni(+2)-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS: Enzyme digestion analysis and sequencing showed that TGF-beta(1) epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass (Mr) of the expressed product by pET28a (+)/CTC was Mr 24,600. The output of the target recombinant protein was approximately 34.8% of the total bacterial protein, mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-beta(1) polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-beta(1) antigenicity and could be used as anti-TGF-beta(1) vaccine. CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF-beta(1) epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-beta(1). The expressed TGF-beta(1) epitope gene shows good immunogenicity and antigenicity.