Identification, characterization, and immunological analysis of complement component 4 from grass carp (Ctenopharyngodon idella).

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp.

Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila.

Aeromonas hydrophila is the causative agent of bacterial septicemia that is frequently observed in grass carp, Ctenopharyngodon idellus. In this study, we evaluated the biological parameters and immune enzymes in the liver of grass carp following A. hydrophila infection and quantified the alterations in liver histology using a semi-quantitative system. For the biological parameters, we found that the liver somatic index (LSI) was more sensitive than Fulton's condition factor (CF) and was significantly decreased at three days post-injection (DPI). At the immune enzyme level, the level of peroxidase (POD) in the liver significantly increased at 1 and 3 DPI. The activity of alkaline phosphatase (ALP) significantly increased at 3 DPI. Similarly, acid phosphatase (ACP) activity significantly increased at 1, 3, and 5 DPI. Histologically, the results indicated that the liver index at 3, 5, and 7 DPI was significantly higher than that of control groups. The regressive alterations as the highly variable reactions patterns and its index at 5 DPI was significantly higher than that of 1, 21 DPI, and the control groups. Based on our results, we suggest that grass carp resist A. hydrophila infection via an innate immune mechanism in the liver. The findings of this study will help elucidate the underlying mechanisms of resistance to A. hydrophila infection.

Mannan-binding lectin-associated serine protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp.

The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.

MiR-26a regulates cell cycle and anoikis of human esophageal adenocarcinoma cells through Rb1-E2F1 signaling pathway.

Resistance to anoikis, the subtype of apoptosis induced by lack of matrix adhesion, contributes to malignant transformation and development of metastasis. MicroRNAs play key regulatory roles in tumorigenesis and metastasis. In this study, we described that miR-26a, which is usually downregulated in tumor cells, is involved in the acquisition of anoikis-resistance of human esophageal adenocarcinoma (EA) cells. Results of qRT-PCR in clinical samples showed that downregulated miR-26a expression is related to tumorigenesis and metastasis of EA. In vitro experiments determined that miR-26a directly participates in the regulation of cell cycle and anoikis of human EA OE33 cells. Further, we identified that Rb1 is the direct functional target of miR-26a, and revealed that the reduction of miR-26a expression leads to increased Rb1 protein level and thus inhibits the function of E2F1, by which it influences the phenotypes of cell cycle and anoikis. The findings we reported here presented the evidence that miR-26a may be involved in regulation of anoikis-resistance of EA cells. Targeting miR-26a may provide a novel strategy to inhibit metastasis.

[Genetic variation analysis of wild populations of grass carp (Ctenopharyngodon idella) using microsatellite markers].

Twelve microsatellites were used to analyze the genetic diversity and genetic structure of eight wild populations of grass carp, among which six populations from Yangtze River (Hanjiang, Wujian, Jiujiang, Shishou, Mudong, and Wanzhou), one population from Pearl River and Heilongjiang River for each, Zhaoqing, and Nenjiang, respectively. Twelve markers showed highly polymorphic and all the eight populations contained high genetic variations. The variations of six populations of Yangtze River and Zhaoqing population of Pearl River were higher than Nenjiang population of Heilongjiang River. Bottleneck analysis revealed that four populations (Zhaoqing, Nenjiang, Mudong, and Wangzhou) had experienced a recent genetic bottleneck, and the effective population size was reduced. Pairwise FST and AMOVA analysis detected significant genetic difference among populations. The pairwise population genetic distances and the UPGMA tree demonstrated that the genetic distances between six populations of Yangtze River and Zhaoqing population were closer and clustered together earlier, as compared to those populations with Nenjiang population. The genetic structure simulation analysis suggested that there were five logic populations of all individuals. The genetic structures of Zhaoqing and Nenjiang populations were shown with independent separation, but the genetic structures of populations from Yangtze River were shown with fuzzy distribution. The high diversity was found in the wild grass carp from three major watersheds in China, which would supply a basis for future genetic improvement. However, the bottleneck effect of some populations should be taken into account in the practical breeding programs.

CpG oligodeoxynucleotides discriminately enhance binding capacity of human naïve B cells to Hepatitis B virus epitopes.

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human naïve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human naïve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human naïve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of naïve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human naïve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed naïve B cells showed the highest binding ability for Env172-180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human naïve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.

Characterization of the intestinal digesta and mucosal microbiome of the grass carp (Ctenopharyngodon idella).

The intestinal microbiome plays a pivotal role in the nutritional digestion and metabolism of the grass carp (Ctenopharyngodon idella). Here, we characterized the digesta and mucosal microbiome of the anterior, middle, and posterior intestine of the grass carp, using 16S rRNA next-generation sequencing. Based on 16S rRNA amplicon data, Proteobacteria, Firmicutes and Bacteroides were the dominant phyla in the intestine of grass carp. Our results also showed that microbial communities of the middle intestine exhibited higher alpha diversity indices compared with the anterior and posterior intestine. The clustering of microbial communities that had either colonized in the digesta or were attached to the mucosa, were significantly tighter in the posterior intestine, based on average unweighted Unifrac distances (P < 0.05). The digesta or mucosa of the anterior and middle intestines were similar in microbial composition, but were significantly different to the posterior intestine (P < 0.05). In digesta and mucosa samples from the posterior intestine, we observed a significantly increased abundance of cellulose-degrading microbiomes, such as Bacteroides, Clostridiales and Spirochaetia (P < 0.05). Our results suggested that the microbiomes of the posterior intestine, either attached to the mucosa or colonized in the digesta, were distinct from the microbiomes of the anterior and middle intestine in grass carp.

16S rRNA sequencing analysis of the correlation between the intestinal microbiota and body-mass of grass carp (Ctenopharyngodon idella).

There appears to be a close correlation between intestinal microbiotas and obesity. Still, our understanding of the relationship between the intestinal microbiota and body-mass in grass carp (Ctenopharyngodon idella) remains limited. Herein, we explored this association in the anterior, middle, and posterior intestine of cohabitating grass carp by using next-generation sequencing of the 16S rRNA gene. The results showed that alpha diversity indices of the low-weight-gain (LWG) groups were higher than that of the high-weight-gain (HWG) groups. HWG groups possessed the decreased ratio of Bacteroidetes to Firmicutes compared with that in the LWG groups. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) revealed that there were significant differences between the HWG and LWG groups. Furthermore, linear discriminant analysis (LDA) coupled with effect size (LEfSe) showed that the order Clostridiales were significantly abundant in the HWG groups. Phylogenetic molecular ecology networks (pMENs) showed a lower average path distance (GD), higher average clustering coefficient (avgCC), and higher average degree (avgK) in the HWG group. Our results suggested that there appeared to be a tight correlation between the intestinal microbiota and body-mass in grass carp. The study provides a referable resource for establishing the relationship between intestinal microbiotas and economic traits, which also lays a foundation for the progress of new fish probiotic in the future.

Agreement of gastroenterologists in traditional Chinese medical differential diagnosis of functional dyspepsia compared with traditional Chinese medical practitioners: A prospective, multicenter study.

OBJECTIVE: To compare the performance of gastroenterologists major in western medicine in diagnosing traditional Chinese medicine (TCM) syndrome types of functional dyspepsia (FD), postprandial distress (PDS) and epigastric pain syndromes (EPS) based on the main symptoms, with that of traditional TCM practitioners in outpatient services. METHODS: Patients with PDS or EPS were enrolled in the study from six tertiary referral centers between January 2016 and December 2017. Their symptoms were first diagnosed by medical doctors, and then by the TCM practitioners. The diagnostic agreement between the gastroenterologists and the TCM practitioners was calculated. The patients' data and their types of FD syndrome were collected and analyzed. RESULTS: In total 160 patients, including 81 with PDS and 79 with EPS were enrolled. The total diagnostic consistency rate between the gastroenterologists and TCM practitioners was 86.3%, while that of PDS and EPS was 85.2% and 87.3%, respectively. The most common type of PDS diagnosed by TCM practitioners was liver-stomach disharmony syndrome (33.3%), spleen deficiency and qi-stagnation syndrome (33.3%), while that for EPS was liver-stomach disharmony syndrome (36.7%). CONCLUSIONS: Gastroenterologists had a high diagnostic agreement about the types of FD syndromes based on differential diagnosis of the main symptoms, compared with TCM practitioners. This may aid gastroenterologists in selecting Chinese medicine for FD-based on syndrome differentiation.

The Diagnosis Performance of the TCM Syndromes of Irritable Bowel Syndrome by Gastroenterologists Based on Modified Simple Criteria Compared to TCM Practitioners: A Prospective, Multicenter Preliminary Study.

PURPOSE: Traditional Chinese medicine (TCM) including Chinese patent medicine has been widely used to treat irritable bowel syndrome (IBS). Syndrome differentiation is the essence of TCM. However, the diagnostic ability of gastroenterologists to detect TCM syndromes in IBS in China remains unknown. The aim of this study was to investigate the ability of gastroenterologists to diagnose the TCM syndromes of IBS based on modified simple criteria compared with TCM practitioners. METHODS: Patients meeting the Rome III criteria for IBS-D or IBS-C were recruited from six tertiary referral centers between January 2016 and December 2017. After learning the diagnosis criteria of the TCM syndromes in IBS, gastroenterologists first diagnosed the syndromes of the enrolled patients. Subsequently, the patients were diagnosed by TCM practitioners. The rate of agreement between the gastroenterologists and TCM practitioners was analyzed. In addition, demographic data and the distribution of TCM syndrome types in IBS were also analyzed. RESULTS: A total of 178 patients (93 males and 85 females), including 131 patients with IBS-D and 47 patients with IBS-C, were enrolled in this study. The rate of agreement of the syndrome diagnosis between the gastroenterologists and TCM practitioners was 84.3%. The diagnosis consistency rates among IBS-D patients and IBS-C patients were 87.0% and 76.5%, respectively. The most common TCM syndrome type in IBS-D patients was liver depression and spleen deficiency syndrome (27.5%), followed by spleen-yang deficiency syndrome (19.8%). Dryness and heat in intestine syndrome was the most common TCM syndrome in IBS-C patients (57.4%). CONCLUSIONS: Gastroenterologists had good diagnostic agreement with TCM practitioners for diagnosing TCM syndrome types in IBS after learning the diagnostic criteria. This knowledge can aid gastroenterologists in selecting suitable Chinese patent medicine to treat IBS.

A Randomized, Double-Blind, Multicenter, Placebo-Controlled Trial of Qi-Zhi-Wei-Tong Granules on Postprandial Distress Syndrome-Predominant Functional Dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a common upper gastrointestinal disorder worldwide, but the current treatments for FD are still unsatisfactory. The aims of this study were to investigate the efficacy and safety of Qi-Zhi-Wei-Tong granules in patients with postprandial distress syndrome (PDS)-predominant FD. METHODS: The study was conducted as a randomized, double-blinded, multicenter, placebo-controlled design in 197 patients with PDS. All participants received placebo treatment for 1 week. Patients whose total symptom score decreased by <50% after the placebo treatment were recruited into the 4-week treatment period, in which they were randomly assigned to be treated with either Qi-Zhi-Wei-Tong granules or placebo. The patients were then followed for 2 weeks without any treatment. Dyspeptic symptoms were scored at weeks 2 and 4 during the random treatment period and 2 weeks after the treatment. Anxiety and depression symptoms were also scored and compared. RESULTS: (1) The total effective rates in the Qi-Zhi-Wei-Tong granules group at weeks 2 and 4 during the random treatment period and 2 weeks after treatment were all significantly higher than those in the placebo group (38.82% vs. 8.75%, P < 0.001; 69.14% vs. 16.25%, P < 0.001; 77.65% vs. 21.25%, P < 0.001). (2) The total dyspeptic symptoms scores in the Qi-Zhi-Wei-Tong granules group at weeks 2 and 4 and 2 weeks after treatment were significantly lower than those in the placebo group. (3) The severity and frequency of each dyspeptic symptom at weeks 2 and 4 and the follow-up period were all significantly lower than those in the placebo group. (4) The anxiety scores in the Qi-Zhi-Wei-Tong granules group were significantly lower than those in the placebo group. (5) Qi-Zhi-Wei-Tong granules did not have more adverse effects than the placebo. CONCLUSION: Qi-Zhi-Wei-Tong granules offer significant symptomatic improvement in PDS with no more adverse effects than placebo. TRIAL REGISTRATION: https://clinicaltrials.gov/, NCT02460601.

Comparison of efficacy and safety of hyoscine butylbromide versus anisodamine for acute gastric or intestinal spasm-like pain: A randomized, double-blinded, multicenter Phase III trial.

OBJECTIVE: Acute gastric or intestinal spasm-like pain is common in clinical setting. Hyoscine butylbromide (HBB), an anti-cholinergic agent, relieves pain in stomach and bowel cramps by inhibiting smooth muscle contractility. In this study, we aimed to compare the efficacy and safety of parenteral HBB and anisodamine for treating acute gastric or intestinal pain. METHODS: In this randomized, controlled, double-blind, parallel-group, multicenter non-inferiority trial, 299 Chinese patients were randomly assigned to HBB or anisodamine in a ratio of 1:1. They were administrated a single dose of either HBB 20 mg or anisodamine 10 mg, and a second dose was given when needed. The primary end-point was the difference in pain intensity (PID) from the pre-dose baseline at 20 min after the first injection. RESULTS: Altogether 295 patients completed the protocol (153 in the HBB and 142 in the anisodamine group). For the primary end-point, the PID was -4.09 (95% confidence interval [CI]: -4.41, -3.76) for the HBB group and -3.66 (95% CI: -4.02, -3.31) for the anisodamine group (P < 0.0001 for non-inferiority). The percentage of patients with at least one adverse event was lower in the HBB group than in the anisodamine group (13.1% vs 17.6%), but there was no statistical significance (P = 0.279). The most frequent adverse events were thirst (7.8%) and dry mouth (2.6%) in the HBB group, and thirst (7.0%), dry mouth (3.5%) and nodal arrhythmia (2.1%) in the anisodamine group. CONCLUSIONS: HBB 20 mg was not inferior to anisodamine 10 mg in pain relief of patients with acute gastric or intestinal spasm-like pain. Both drugs were safe and well tolerated.

Intravenous Esomeprazole for Prevention of Peptic Ulcer Rebleeding: A Randomized Trial in Chinese Patients.

INTRODUCTION: High-dose intravenous esomeprazole is the only approved pharmacological treatment for the prevention of peptic ulcer rebleeding (currently approved in over 100 countries worldwide), but has not yet been approved in China. This study aimed to evaluate a high-dose esomeprazole intravenous regimen vs. an active control (cimetidine) for the prevention of rebleeding in Chinese patients with a high risk of peptic ulcer rebleeding who had undergone primary endoscopic hemostatic treatment. METHODS: This was a parallel-group study conducted at 20 centers in China. The study comprised a randomized, double-blind, intravenous treatment phase of 72 h in which 215 patients received either high-dose esomeprazole (80 mg + 8 mg/h) or cimetidine (200 mg + 60 mg/h), followed by an open-label oral treatment phase in which all patients received esomeprazole 40 mg tablets once daily for 27 days. The primary outcome was the rate of clinically significant rebleeding within the first 72 h after initial endoscopic hemostatic therapy. Secondary outcomes included the rates of clinically significant rebleeding within 7 and 30 days; proportions of patients who had endoscopic retreatment and other surgery due to rebleeding; and number of blood units transfused. RESULTS: The rate of clinically significant rebleeding within 72 h was low overall (3.3%) and numerically lower in patients treated with esomeprazole compared with cimetidine (0.9% vs. 5.6%). Overall, the results of the secondary outcomes also showed a numerical trend towards superiority of esomeprazole over cimetidine. All treatments were well tolerated. CONCLUSION: In this phase 3, multicenter, randomized trial conducted in China, esomeprazole showed a numerical trend towards superior clinical benefit over cimetidine in the prevention of rebleeding in patients who had successfully undergone initial hemostatic therapy of a bleeding peptic ulcer, with a similar safety and tolerability profile. These findings suggest that esomeprazole may be an alternative treatment option to cimetidine for this indication in China. FUNDING: AstraZeneca. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT01757275.

Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

Nuclear and mitochondrial DNA microsatellite instability in hepatocellular carcinoma in Chinese.

AIM: To study the nuclear microsatellite instability (nMSI) at BAT26 and mitochondrial microsatellite instability (mtMSI) in the occurrence and development of hepatocellular carcinoma and the relationship between nMSI and mtMSI. METHODS: nMSI was observed with PCR and mtMSI with PCR-SSCP in 52 cases of hepatocellular carcinoma. RESULTS: mtMSI was detected in 11 out of the 52 cases of hepatocellular carcinoma (21.2%). Among the 11 cases of hepatocellular carcinoma with mtMSI, 7 occurred in one locus and 4 in 2 loci. The frequency of mtMSI in the 52 cases of hepatocellular carcinoma showed no correlation to sex, age, infection of hepatitis B, liver cirrhosis as well as positive AFP of the patients (P>0.05). In addition, nMSI was detected in 3 out of 52 cases of hepatocellular carcinoma (5.8%) and there was no correlation of the incidence of mtMSI to that of nMSI (P>0.05). CONCLUSION: mtMSI may be involved in the coccurrence and development of hepatocellular carcinoma and it is independent of nMSI.

Mitochondrial microsatellite instability in gastric cancer and its precancerous lesions.

AIM: To evaluate the role of mitochondrial microsatellite instability (mtMSI) in gastric carcinogenesis. METHODS: MtMSI was measured with PCR-single strand conformation polymorphism (PCR-SSCP) in 68 cases of advanced gastric cancer, 40 cases of chronic gastritis, 30 cases of intestinal metaplasia and 20 cases of dysplasia. RESULTS: MtMSI was observed in 12.5% (5 of 40) of chronic gastritis, 20.0% (6 of 30) of intestinal metaplasia, 25.0% (5 of 20) of dysplasia and 38.2% (26 of 68) of gastric cancer. These findings showed a sequential accumulation of mtMSI in the histological progression from chronic gastritis to gastric cancer. An association of mtMSI with intestinal histological type and distal location was found (P=0.001 and P=0.002), whereas no significant correlation was found between mtMSI and age at diagnosis, sex, tumor size, depth of invasion, lymph node spread and clinical stages (P>0.05). CONCLUSION: MtMSI may play an early and important role in the gastric carcinogenesis pathway, especially in the intestinal type and distal gastric cancer.

Effect of NF-kappaB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand.

AIM: To study the effect of NF-kappaB, survivin, Bcl-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells. METHODS: Gastric cancer cells of SGC-7901, MKN28, MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis inducing ligand (TRAIL) with a flow cytometer. The expression of NF-kappaB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot. RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45, AGS and SGC-7901cells respectively. Western blot revealed that the expressions of NF-kappaB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells. CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines. The anticancer potency of TRAIL is associated with the decreased expression of NF-kappaB and survivin and increased expression of Caspase3 of gastric cancer cells.

Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability.

AIM: To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers. METHODS: Mutation of hMLH1 was detected by Two-dimensional electrophoresis (Two-D) and DNA sequencing; Methylation of hMLH1 promoter was measured with methylation-specific PCR; MSI was analyzed by PCR-based methods. RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern. Methylation of hMLH1 promoter was detected in 11(16.2 %) gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25 %) of the tumors analyzed. Three hMLH1 mutations were all detected in MSI-H (>=2 loci, n=8), but no mutation was found in MSI-L (only one locus, n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5 %, 7/8) was significantly higher than that in MSI-L (11.1 %, 1/9) or MSS (5.9 %, 3/51) (P<0.01-0.001), but no difference was found between MSI-L and MSS (P>0.05). CONCLUSION: Both mutation and methylation of hMLH1 are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.

Effect of Helicobacter pylori infection on expression of Bcl-2 family members in gastric adenocarcinoma.

AIM: To investigate the effect of Helicobacter pylori (H pylori) infection on the expressions of Bcl-2 family members in gastric adenocarcinoma. METHODS: Gastric adenocarcinoma and resection margin tissues of 95 patients were studied. Semi-quantitative RT-PCR was used to measure Bid, Bax and Bcl-2 mRNA expressions. RESULTS: Expressions of Bid and Bax in gastric adenocarcinoma tissues without H pylori infection, with cagA- H pylori infection and cagA+ H pylori infection increased significantly in turn (Bid, 0.304, 0.422 and 0.855 respectively, P<0.05; Bax, 0.309, 0.650 and 0.979 respectively, P<0.05). Bcl-2 mRNA levels increased significantly in gastric adenocarcinoma tissues with cagA- H pylori infection and cagA+ H pylori infection, compared with those without H pylori infection (0.696 and 0.849 vs 0.411, P<0.05). Expressions of Bid, Bax and Bcl-2 in resection margin tissues without H pylori infection, with cagA- H pylori infection and cagA+ H pylori infection increased significantly in turn (Bid, 0.377, 0.686 and 0.939 respectively, P<0.05; Bax, 0.353, 0.645 and 1.001 respectively, P<0.05; Bcl-2, 0.371, 0.487 and 0.619 respectively, P<0.05). In H pylori negative specimens, expressions of Bid and Bax correlated negatively with that of Bcl-2 respectively in adenocarcinoma tissues (Bid vs Bcl-2, r=-0.409, P<0.05; Bax vs Bcl-2, r=-0.451, P<0.05). In H pylori positive specimens, expressions of Bid and Bax did not correlate with that of Bcl-2 in adenocarcinoma tissues (Bid vs Bcl-2, r=0.187, P>0.05; Bax vs Bcl-2, r=0.201, P>0.05), but correlated positively with that of Bcl-2 respectively in resection margin tissues (Bid vs Bcl-2, r=0.331, P<0.05; Bax vs Bcl-2, r=0.295, P<0.05). CONCLUSION: H pylori may enhance Bid, Bax and Bcl-2 mRNA levels and cause deregulation of these apoptosis-associated genes expressions, which may play a role during development of gastric adenocarcinoma induced by H pylori.

Effects of surface gastric pacing on gastric myoelectrical activity and plasma motilin in a canine model of gastric motility disorders.

OBJECTIVE: To investigate the effects of surface gastric pacing on gastric myoelectrical activity and plasma motilin concentration in a canine model of gastric motility disorders. METHODS: Ten healthy mongrel dogs were divided into two groups: an experimental group of six dogs and control group of four dogs. The model of gastric motility disorders was established in the experimental group with truncal vagotomy combined with injection of glucagon. Gastric serosal myoelectrical activity was recorded with a four-channel computer analysis device. Plasma motilin concentration was measured with a radioimmunoassay (RIA) kit. Surface gastric pacing at 1.1-1.2-fold the intrinsic slow-wave frequency superimposed with a series of high frequency pulses (10-30 Hz) was performed for 45 min daily for 1 month in the conscious dogs. RESULTS: The basic electrical rhythm (BER) amplitude (2.32 +/- 0.35 mV) and propagation velocity (4.06 +/- 0.40 cm/s) of the dogs with bilateral truncal vagotomy in the fed state decreased more significantly than those of the controls (4.25 +/- 0.12 mV, 6.92 +/- 0.24 cm/s) (P < 0.03). After long-term surface gastric pacing, the BER amplitude (3.97 +/- 0.19 mV) and propagation velocity (5.57 +/- 0.48 cm/s) was increased significantly compared with before pacing (P < 0.05). Postprandial gastric dysrhythmias were provoked by large doses of glucagon; the percentage of regular slow waves of the dogs with vagotomy was markedly reduced from 67.4 +/- 6.2% at baseline to 10.0 +/- 6.7% (P < 0.001), and that of the control was also decreased from 87.1 +/- 6.9% to 35.0 +/- 11.0% (P < 0.01), but the entrainment of gastric slow waves was 100% by means of gastric pacing at optimal parameters. There was a significant increase in the plasma concentration of motilin at the phase III of the interdigestive myoelectrical complex (IMC III) in the dogs with bilateral truncal vagotomy (baseline vs vagotomy, 184.29 +/- 9.81 pg/mL vs 242.09 +/- 17.22 pg/mL; P< 0.01). However, the plasma motilin concentration (212.55 +/- 11.20 pg/mL; P < 0.02) was decreased significantly after long-term gastric pacing. Before gastric pacing the plasma motilin concentration showed an equally negative correlation with the BER amplitude, and propagation velocity in the dogs with vagotomy in the fed state (r = -0.473, r = -0.807, P < 0.04), but after long-term gastric pacing, the plasma motilin concentration showed an equally positive correlation with the BER amplitude and propagation velocity (r = 0.523, r = 0.896, P < 0.02). CONCLUSIONS: Surface gastric pacing with optimal pacing parameters is able to entrain completely propagated slow waves, improve the parameters of gastric myoelectrical activity and normalize gastric dysrhythmias induced by a pharmacological agent. Surface gastric pacing might be useful in the treatment of gastric dysrhythmia. The gastric myoelectrical activity correlated well with the plasma motilin concentration before and after pacing, which suggests that motilin could modulate the effect of gastric pacing through alteration of the gastric myoelectrical parameters.