两种检测急性冠脉综合征患者CKMB及CTNI方法一致性分析

目的:评价急性冠脉综合征患者中,化验室心肌酶及标志物分析法检测心肌肌酸激酶同工酶(CKMB)和心肌肌钙蛋白I(CTNI)结果与心梗三联床旁检测法是否一致及其一致性程度。方法:对78例临床诊断急性冠脉综合征的患者同时采集两份标本,分别应用化验室心肌酶及标志物分析法和心梗三联床旁检测法进行CKMB及CTNI测定。采用配对卡方检验(McNemar Test)和一致性检验(Kappa检验)方法进行统计学分析。结果:两种方法测得CKMB和CTNI值比较差异无统计学意义(t=1.123,P=0.265;t=-0.326,P=0.747),且一致性较好(Kappa=0.579,P<0.05;Kappa=0.546,P<0.05)。采用配对卡方检验结果显示,两种方法测得CKMB和CTNI值升高程度差异均无统计学意义(P>0.05)。结论:化验室心肌酶及标志物分析法与心梗三联床旁检测法检测CKMB及CTNI值结果一致性较好。

Prominent role of y-glutamyl-transpeptidase on the growth of Helicobacter pylori

AIM: γ-glutamyl transpeptidase (GGT) has been reported as a virulence and colonizing factor of Helicobacter pylori (Hpylori). This study examined the effect of GGT on thegrowth of H pylori. METHODS: Standard H pylori strain NCTC 11637 and 4 dinical isolates with different levels of GGT activity as measured by an enzymatic assay were used in this study. Growth inhibilJon and stimulation studies were carried out by culturing H pyloriin brain heart infusion broth supplemented with specific GGT inhibitor (L-serine sodium borate complex, SBC) or enhancer (glutathione together with glycyl-glycine), respectively. The growth profiles of Hpyloriwere determined based on viable bacterial count at time interval. RESULTS: Growth was more profuse for Hpyloriisolates with higher GGT activity than those present with lower GGT activity. However, in the presence of SBC, growth of Hpylori was retarded in a dose dependent manner (P = 0.034). In contrast, higher growth rate was observed when GGT activity was enhanced in the presence of glutathione and glycyl-glycine. CONCLUSION: Higher GGT activity provides an advantage to the growth of Hpy/oriin vitro. Inhibition of GGT activity by SBC resulted in growth retardation. The study shows that GGT plays an important role on the growth of Hpy/ori.

Effect of TYLCV Infection on Leaf Anatomical Structure and Protective Enzyme System of Tomato

[Objective] The aim was to study the effect of tomato yellow leaf curl virus （TYLCV） infection on leaf anatomical structure and protective enzyme system of tomato. [Method] The anatomical structure of infected and healthy leaves of tomato were observed and compared by using paraffin section method. The activity changes of SOD, POD and CAT in the infected leaves of tomato were determined. [ Result] The results revealed that there were some differences in anatomical structure between healthy and infected leaves. Some cells of infected leaves were damaged so that the leaves curled and became yellow, which affected the normal function of organs. Compared with control, enzyme activities in the tomato plants infected by TYLCV were enhanced at the early periods and higher than that in control, then started to decline at the middle and late periods but lower than that in control.[ Conclusion] After infection by TYLCV, the leaf anatomical structure of tomato was changed greatly and the protective enzyme system was damaged severely, and affected the normal physJological metabolic functions of tissues and organs in tomato in further.

Clinical value of fecal calprotectin in determining disease activity of ulcerative colitis

AIM： To investigate possibility and clinical application of fecal calprotectin in determining disease activity of ulcerative colitis （UC）. METHODS： The enzyme-linked immunosorbent assay （ELISA） was used to measure the concentrations of calprotectin in feces obtained from 66 patients with UC and 20 controls. C-reactive protein （CRP）, erythrocyte sedimentation rate （ESR）, acid glycoprotein （AGP） were also measured and were compared with calprotectin in determining disease activity of UC. The disease activity of UC was also determined by the Sutherland criteria. RESULTS： The fecal calprotectin concentration in the patients with active UC was significantly higher than that in the inactive UC and in the controls （402.16 ± 48.0 μg/g vs 35.93 ± 3.39 μg/g, 11.5 ± 3.42 μg/g, P 〈 0.01）. The fecal calprotectin concentration in the inactive UC group was significantly higher than that in the control group （P 〈 0.05）. A significant difference was also found in the patients with active UC of mild, moderate and severe degrees. The area under the curve of the receiver operating characteristics （AUCR^c） was 0.975, 0.740, 0.692 and 0.737 for fecal calprotectin, CRP, ESR and AGP, respectively. There was a strong correlation between the fecal calprotectin concentration and the endoscopic gradings for UC （r = 0.866, P 〈 0.001）. CONCLUSION： Calprotectin in the patient＇s feces can reflect the disease activity of UC and can be used as a rational fecal marker for intestinal inflammation in clinical practice. This kind of marker is relatively precise, simple and noninvasive when compared with other commonlyused markers such as CRP, ESR and AGP.

Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ 11 dextranase mouthwash

We adopted the response surface methodology using single factor and orthogonal experiments to optimize four types of antimicrobial agents that could inhibit biofilm formation by Streptococcus mutans, which is commonly found in the human oral cavity and causes tooth decay. The objective was to improve the function of marine Arthrobacter oxydans KQll dextranase mouthwash （designed and developed by our laboratory）. The experiment was conducted in a three-level, four-variable central composite design to determine the best combination of ZnSO4, lysozyme, citric acid and chitosan. The optimized antibacterial agents were 2.16 g/L ZnSO4, 14 g/L lysozyme, 4.5 g/L citric acid and 5 g/L chitosan. The biofilm formation inhibition reached 84.49%. In addition, microscopic observation of the biofilm was performed using scanning electron microscopy and confocal laser scanning microscopy. The optimized formula was tested in marine dextranase Arthrobacter oxydans KQ11 mouthwash and enhanced the inhibition of S. mutans. This work may be promoted for the design and development of future marine dextranase oral care products.

Effect of synbiotics on intestinal microflora and digestive enzyme activities in rats

AIM: To investigate the effect of synbiotics, i.e. probiotics and prebiotics mixture, on the gut microbial ecology and digestive enzyme activities in rats. METHODS: Forty-eight SD rats weighing about 280 g were used in this study. Rats were divided into three groups according to the contents of probiotics and prebiotics mixture in the feed as control, low and high dose groups. The duration of the experiment was 8 wk. RESULTS: Compared with the control group, thefecal Lactobacillus and Bifidobacterium counts were significantly increased and the fecal Coliform organism counts were markedly reduced in the low and high dose groups. Concerning the digestive enzyme activity of jejunum, only lactase activity increased in low dose group. However, significant increase of lipase, lactase, sucrase, and isomaltase activities were observed in high dose group.CONCLUSION: Intake of low and high dosages of probiotics and prebiotics mixture significantly improved the ecosystem of the intestinal tract by increasing the probiotics population and digestive enzyme activities in rats.

Measurement of serum paraoxonase-1 activity in the evaluation of liver function

Paraoxonase-1 (PON1) is an esterase and lactonase synthesized by the liver and found in the circulation associated with high-density lipoproteins. The physiological function of PON1 seems to be to degrade specific oxidized cholesteryl esters and oxidized phospholipids in lipoproteins and cell membranes. PON1 is, therefore, an antioxidant enzyme. Alterations in circulating PON1 levels have been reported in a variety of diseases involving oxidative stress including chronic liver diseases. Measurement of serum PON1 activity has been proposed as a potential test for the evaluation of liver function. However, this measurement is still restricted to research and has not been extensively applied in routine clinical chemistry laboratories. The reason for this restriction is due to the problem that the substrate commonly used for PON1 measurement, paraoxon, is toxic and unstable. The recent development of new assays with non-toxic substrates makes this proposal closer to a practical development. The present editorial summarizes PON1 biochemistry and function, its involvement with chronic liver impairment, and some aspects related to the measurement of PON1 activity in circulation.

Effect of Bioactive Peptides(BPs) on the Development of Pacific White Shrimp(Litopenaeus vannamei Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal(FM) with bioactive peptides(BPs) in diet of white shrimp(Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0%(control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate(WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con(P < 0.05), while no significant difference was found on survival rate and feed conversion ratio(FCR); 2) the whole-body crude protein(CP) and crude lipids(CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase(ACP), lysozyme(LZM), superoxide dismutase(SOD), phenol oxidase(PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in p H and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.

Gastric cancer cells induce human CD4^+ Foxp3^+ regulatory T cells through the production of TGF-β1

AIM: To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer. METHODS: The frequencies of CD4 + Foxp3 + Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer byflow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the coculture of gastric cancer cell line, MGC-803, with sorting CD4 + T cells. The normal gastric mucosa cell line, GES-1, was used as the control. The production of TGF-β1 was detected in supernatant of MGC and GES-1. The carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay was performed to evaluate the proliferation characteristics of induced Tregs. Neutralizing anti-TGF-β1 antibody was added to the co-culture system for neutralization experiments. RESULTS: The level of serum TGF-β1 in gastric cancer patients (15.1 ± 5.5 ng/mL) was significantly higher than that of the genderand age-matched healthy controls (10.3 ± 3.4 ng/mL) (P < 0.05). Furthermore, the higher TGF-β1 level correlated with the increased population of CD4 + Foxp3 + Tregs in advanced gastric cancer (r = 0.576, P < 0.05). A significant higher frequency of CD4 + Foxp3 + Tregs was observed in PBMCs cultured with the supernatant of MGC than GES-1 (10.6% ± 0.6% vs 8.7% ± 0.7%, P < 0.05). Moreover, using the purified CD4 + CD25 T cells, we confirmed that the increased Tregs were mainly induced from the conversation of CD4 + CD25 naive T cells, and induced Tregs were functional and able to suppress the proliferation of effector T cells. Finally, we demonstrated that gastric cancer cells induced the increased CD4 + Foxp3 + Tregs via producing TGF-β1. Gastric cancer cells upregulated the production of TGF-β1 and blockade of TGF-β1 partly abrogated Tregs phenotype. CONCLUSION: Gastric cancer cell can induce Tregs development via producing TGF-β1, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses.

Expression of tissue inhibitor of matrix metalloproteinases-1 during aging in rat liver

AIM: To investigate the expression and role of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) during natural aging in rat liver and to detect the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. METHODS: The rats were divided into 3-mo-old group (n=5), 10-mo-old group (n=5) and 24-mo-old group (n=5). Histopathologic changes of liver were observed with HE and Masson stain. The location and protein expressions of TIMP-1 were determined by immunohistochemistry and Western blot; message RNA (mRNA) levels were measured in livers from rats of various ages by semi-quantitative reverse transcriptional polymerase chain reaction (RT PCR). In addition, the expression of MMP-2 and MMP-9 was assessed by RT-PCR and Western blot. RESULTS: Histologic examination showed that the aging liver had excessive fatty degeneration and collagen deposition. Immunohistochemical staining showed that TIMP-1 related antigen in livers was located in cytoplasm. The protein expression of TTMP-1 was significantly higher in the oldest animals and the mRNA expression was increased significantly in the 24-mo-old rats (t= 4.61, P= 0.002<0.05, 24-vs 10-mo-old rats; t=4.31, P=0.003<0.05, 24- vs 3-mo-old rats). The expression of MMP-2 and MMP-9 had no change during aging; the ratios TIMP-1/MMP-2 and TIMP-1/MMP-9 in aging liver were significantly higher than those in maturation and young livers. CONCLUSION: TIMP-1 may play an important role in the process of liver aging.

Phenotypic and Genotypic Comparison of Pseudomonas stutzeri in Freshwater Fish in Indonesia

Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （Jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA） （Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP） （Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （Jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of Jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.

Enhanced Production of Hybrid Extracellular β-Glucanase by Re-combinant Escherichia coli Using Experimental Design Method

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid ex-tracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those ob-tained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.

Use of butyrate or glutamine in enema solution reduces inflammation and fibrosis in experimental diversion colitis

AIM:To investigate whether butyrate or glutamine enemas could diminish inflammation in experimental diversion colitis.METHODS:Wistar specific pathogen-free rats were submitted to a Hartmann's end colostomy and treated with enemas containing glutamine,butyrate,or saline.Enemas were administered twice a week in the excluded segment of the colon from 4 to 12 wk after the surgical procedure.Follow-up colonoscopy was performed every 4 wk for 12 wk.The effect of treatment was evaluated using video-endoscopic and histologic scores and measuring interleukin-1β,tumor necrosis factor-alpha,and transforming growth factor beta production in organ cultures by enzyme linked immunosorbent assay.RESULTS:Colonoscopies of the diverted segment showed mucosa with hyperemia,increased number of vessels,bleeding and mucus discharge.Treatment with either glutamine or butyrate induced significant reductions in both colonoscopic(P < 0.02) and histological scores(P < 0.01) and restored the densities of collagen fibers in tissue(P = 0.015;P = 0.001),the number of goblet cells(P = 0.021;P = 0.029),and the rate of apoptosis within the epithelium(P = 0.043;P = 0.011) to normal values.The high levels of cytokines in colon explants from rats with diversion colitis significantly decreased to normal values after treatment with butyrate or glutamine.CONCLUSION:The improvement of experimental diversion colitis following glutamine or butyrate enemas highlights the importance of specific luminal nutrients in the homeostasis of the colonic mucosa and supports their utilization for the treatment of human diversion colitis.

Proteinases from the digestive organs of black carp （Mylopharyngodon piceus）： Partial characterization and protein digestibility in vitro

A series of experiments based on electrophoretical and biochemical assays were conducted to partially characterize proteinases present in the hepatopancreas and intestine of black carp （Mylopharyngodon piceus）, and investigate enzymatic activity and protein digestibility in vitro. Casein digestion assays revealed the presence of acidic proteinases with optimum activity in the range of pH 2.0-2.5 and alkaline proteinases with significantly higher activities both in the range of pH 8.1-8.6 and near pH 9.5. The inhibition and substrate specificity assays showed that trypsin and chymotrypsin are the main active components of the alkaline proteinases. The SDS-substrate-PAGE showed that the crude extract of black carp intestine had eight types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa while the crude extract of black carp hepatopancreas had six types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa. These enzymes were characterized as trypsin （27.5 kDa, 30.1 kDa）, chymotrypsin （40.5 kDa, 42.5 kDa）, serine proteinases （32.1 kDa, 33.2 kDa） and non-serine proteinase （61.5 kDa, 78.5 kDa）.In vitro protein digestibility assays showed that black cardcan be able to utilize a wider range of proteins.

Serum level of galectin-3 and its clinical significance in patients with chronic Keshan disease

Keshan disease （KD） is a fatal endemic dilated cardiomyopathy with unclear etiology and pathogenesis, and a high mortality in China. Pathologic studies confna＇ned that different degree of myocardial fibrosis existed in various types of KD. Myocardial fibrosis is an important contributor to the pathophysiology of left ventricular remodeling. Recently, galectin-3 （Gal-3） as a marker of cardiac fibrosis and heart failure was approved by the US FDA. The aim of this study was to evaluate the changes of serum level of Gal-3 in chronic KD （CKD） mad their clinical implications. Methods： The levels of serum Gal-3 were measured by using enzyme-linked immunosorbent assay （ELISA） in 37 CKD patients and 32 healthy controls. Echocardiography was used to determine parameter of left ventricular ejection fraction （LVEF） and left ventricular end-diastolic dimension （LVEDD）. Results： The serum concentration of Gal-3 （[95.81：i：18.99] ng/ml versus [48.16-＋11.09] ng/ml, t=6,906, P〈0.001） and LVEDD （[60.46＋7.63] mm versus （42.69^-10.66） ram, t=3.61, P〈0.01） were significantly higher, while LVEF （[42.69-J：10.661% versus [62.16~6.381%, t=4.679, P〈0.01） were significantly lower in CKD patients compared with healthy controls. A negativecorrelation was found between elevated Gal-3 and lower LVEF （r=--0.882, P〈0.001） and a positive correlation was found between elevated Gal-3 levels and enlarged LVEDD （r=0.834, P〈0.001） or higher NYHA class （r=0.854, P〈0.01） in CKD patients. Conclusion： Serum concentration of Gal-3 is strongly correlated with poorer left ventricular systolic function, enlarged LVEDD and higher New York Heart Association （NYHA） class, which may provide indirectly diagnostic information on myocardial fibrosis and heart function in CKD patients

Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis.Expression of α-smooth muscle actin (α-SMA) and collagen typesⅠand Ⅲ was evaluated by real-time PCR.Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method.Finally,collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay.RESULTS:The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen.siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner.Moreover,HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs.CONCLUSION:This study suggests a significant functional role for HMGB1 in the development of liver fibrosis.It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

Nitric oxide-releasing aspirin but not conventional aspirin improves healing of experimental colitis

AIM:To determine the effect of non-selective cyclooxygenase (COX) inhibitors,selective COX-2 inhibitors and nitric oxide (NO)-releasing aspirin in the healing of ulcerative colitis.METHODS:Rats with 2,4,6 trinitrobenzenesulfonic acid (TNBS)-induced colitis received intragastric (ig) treatment with vehicle,aspirin (ASA) (a nonselective COX inhibitor),celecoxib (a selective COX-2 inhibitor) or NO-releasing ASA for a period of ten days.The area of colonic lesions,colonic blood flow (CBF),myeloperoxidase (MPO) activity and expression of proinflammatory markers COX-2,inducible form of nitric oxide synthase (iNOS),IL-1β and tumor necrosis factor (TNF)-α were assessed.The effects of glyceryl trinitrate (GTN),a NO donor,and 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide,onopotassium salt (carboxy-PTIO),a NO scavenger,administered without and with ASA or NO-ASA,and the involvement of capsaicin-sensitive afferent nerves in the mechanism of healing the experimental colitis was also determined.RESULTS:Rats with colitis developed macroscopic and microscopic colonic lesions accompanied by a significant decrease in the CBF,a significant rise in colonic weight,MPO activity and plasma IL-1β and TNF-α levels.These effects were aggravated by ASA and 5-(4-chlorophenyl)-1-(4-methoxyphenyl)3-(trifluoromethyl)-1H-pyrazole (SC-560),but not celecoxib and counteracted by concurrent treatment with a synthetic prostaglandin E 2 (PGE 2) analog.Treatment with NO-ASA dose-dependently accelerated colonic healing followed by a rise in plasma NO x content and CBF,suppression of MPO and downregulation of COX-2,iNOS,IL-1β and TNF-α mRNAs.Treatment with GTN,the NO donor,significantly inhibited the ASA-induced colonic lesions and increased CBF,while carboxy-PTIO or capsaicin-denervation counteracted the NO-ASAinduced improvement of colonic healing and the accompanying increase in the CBF.These effects were restored by co-treatment with calcitonin gene related peptide (CGRP) and NO-ASA in capsaicin-denervated animals.CONCLUSION:NO-releasing ASA,in contrast to ASA,COX-1 inhibitors,and SC-560,accelerated the healing of colitis via a mechanism involving NO mediated improvement of microcirculation and activation of sensory nerves releasing CGRP.

RNF13： a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin ligase activity. Immunohistochemical analysis of pancreatic ductal adenocarcinoma （PDAC） and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples （41.7%, 30/72）, and its expression was significantly associated with histological grading （P= 0.024）. In addition, RNF13 was detected in precancerous lesions： tubular complexes in chronic pancreatitis （CP） and pancreatic intraepithelial neoplasia （PanIN） （79.3%, 23/29 and 62.8%, 22/35, respectively）. Moreover, RNF13 staining was significantly correlated with Tenascin-C expression （P = 0.004） in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells

AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase(hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.