In silico analysis and verification of S100 gene expression in gastric cancer.

BACKGROUND: The S100 protein family comprises 22 members whose protein sequences encompass at least one EF-hand Ca2+ binding motif. They were involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. However, the expression status of S100 family members in gastric cancer was not known yet. METHODS: Combined with analysis of series analysis of gene expression, virtual Northern blot and microarray data, the expression levels of S100 family members in normal and malignant stomach tissues were systematically investigated. The expression of S100A3 was further evaluated by quantitative RT-PCR. RESULTS: At least 5 S100 genes were found to be upregulated in gastric cancer by in silico analysis. Among them, four genes, including S100A2, S100A4, S100A7 and S100A10, were reported to overexpressed in gastric cancer previously. The expression of S100A3 in eighty patients of gastric cancer was further examined. The results showed that the mean expression levels of S100A3 in gastric cancer tissues were 2.5 times as high as in adjacent non-tumorous tissues. S100A3 expression was correlated with tumor differentiation and TNM (Tumor-Node-Metastasis) stage of gastric cancer, which was relatively highly expressed in poorly differentiated and advanced gastric cancer tissues (P < 0.05). CONCLUSION: To our knowledge this is the first report of systematic evaluation of S100 gene expressions in gastric cancers by multiple in silico analysis. The results indicated that overexpression of S100 gene family members were characteristics of gastric cancers and S100A3 might play important roles in differentiation and progression of gastric cancer.

[Laparoscopy-assisted D2 total gastrectomy in advanced gastric cancer].

OBJECTIVE: To investigate the efficacy and advantages of laparoscopy-assisted total gastrectomy (LATG) with D2 dissection of lymph nodes versus conventional open D2 total gastrectomy (OTG) in advanced gastric cancer. METHODS: One hundred and twenty-five patients with advanced gastric cancer in the middle or upper third of the stomach were operated on from July 2005 to March 2007. Of the patients, 59 cases received LATG and 66 OTG with D2 lymph nodes dissection. Clinical data were recorded and compared between the two groups. RESULTS: No patient in the LATG group converted to conventional operation with laparotomy. No operation mortality and no severe morbidity occurred in LATG group. As compared with OTG group, in LATG group operation time was longer [(330 +/- 71) min vs. (261 +/- 54) min, P =0.005] in LATG group, but with similar number of lymph node retrieval (36 +/- 13 vs. 34 +/- 16, P =0.450), less operation blood loss [(175 +/- 101) ml vs. (359 +/- 210) ml, P =0.003], earlier recovery of bowel activity (P = 0.015), and a shorter duration of fever after operation (P = 0.024). CONCLUSIONS: LATG with D2 lymph node dissection in advanced gastric cancer is safe and technically feasible with better operative access and visual field, less operation blood loss and earlier recovery.

Right ventricular outflow tract septal pacing versus apical pacing: A prospective, randomized, single-blind 5-years follow-up study of ventricular lead performance and safety.

Lead placement for ventricular pacing variably impacts the physiological benefit of the patient. This study evaluated the ventricular lead performance and safety of right ventricular outflow tract septal pacing in patients with bradyarrhythmia in South China over 60-month follow-up. Totally, 192 patients (108 males, and 84 females, 63±21 years old) with bradyarrhythmia were randomly divided into two groups. The right ventricular outflow tract septum (RVOTs) group had lead placement near the septum (n=97), while the right ventricular apex (RVA) group had a traditional apical placement (n=95). RV septal lead positioning was achieved with a specialized stylet and confirmed using fluoroscopic projection. All patients were followed up for 60 months. Follow-up assessment included stimulation threshold, R-wave sensing, lead impedance and lead complications. The time of electrode implantation in both the ROVTs and RVA groups were significantly different (4.29±0.61 vs. 2.16±0.22 min; P=0.009). No differences were identified in threshold, impedance or R-wave sensing between the two groups at 1st, 12th, 36th and 60th month during the follow-up period. No occurrence of electrode displacement, increased pacing threshold or inadequate sensing was found. The long-term active fixation ventricular electrode performance in RVOTs group was similar to that in RVA group. RVOTs pacing near the septum using active fixation electrodes may provide stability during long-term follow-up period.

Accelerated acute allograft rejection accompanied by enhanced T-cell proliferation and attenuated Treg function in RBP-J deficient mice.

Acute allograft rejection (AAR) involves both the innate and the adaptive immune systems. As a critical pathway in peripheral T-cell differentiation and function, Notch signaling is potentially involved in the modulation of AAR, but its role in alloimmune responses has not been fully addressed. By using fully MHC-mismatched allograft transplantation model and T-cell specific RBP-J deficient mice, we examined the role of Notch/RBP-J pathway in alloimmune responses in vivo. AAR was significantly accelerated in RBP-J deficient mice compared with the wild-type controls, as demonstrated by the marked reduction in graft survival. The reduction in graft survival was associated with augmented alloantigen specific T-cell proliferation and increased number of Th1, Th2, and Th17 cells in the RBP-J deficient recipient mice. Furthermore, although the frequency of CD4(+)CD25(+)Foxp3(+) Tregs was intact in RBP-J knockout recipients, their ability to suppress Teff responses in vitro was significantly dampened. These findings suggest that Notch/RBP-J pathway may attenuate AAR by suppressing in vivo expansion of alloreactive T-cell proliferation and facilitating CD4(+)CD25(+) Treg suppression ability, indicating that Notch pathway could be exploited to limit T-cell-mediated AAR.

[Relationship of angiotensin-converting enzyme 2 gene polymorphism with the prognosis of hypertensive stroke patients].

OBJECTIVE: To study the relationship between angiotensin-converting enzyme 2 (ACE2) gene G9570A polymorphisms and the clinical outcome of stroke patients with essential hypertension (EH) in South China Han population. METHOD: The ACE2 gene polymorphisms were detected in 141 stroke patients with EH and 156 patients with EH using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The genetic marker was tested for its association with the baseline measurements and clinical outcomes of the patients over a median follow-up period of 22 months. As the ACE2 gene is X-linked, analyses were performed for male and female patients separately. RESULTS: The A allele frequency in the stroke patients was significantly different from that in the EH patients, and the AA allele frequency in the female patients was significantly different between the two groups (P<0.01). Kaplan-Meier model analysis showed that ACE2 gene polymorphism was not associated with the the patients' prognosis (P>0.05). Multivariate Cox's proportional hazard regression model identified age (RR=1.057, 95%CI: 1.020, 1.095), blood glucose (RR=1.575, 95%CI: 1.178, 2.104), hypertriglyceridemia (RR=1.947, 95%CI: 1.503, 2.780), blood creatinine (RR=1.034, 95%CI: 1.001, 1.068), and blood uric acid (RR=1.056, 95%CI: 1.002, 1.097) as the risk factors associated with the mortality. CONCLUSION: Stroke occurs more likely in hypertensive patients carrying the A/AA allele than those carrying other alleles. The ACE2 gene G9570A polymorphisms may be associated with the occurrence of stroke in EH patients in South China, but may not have a strong correlation to the prognosis.

[Correlation of angiotensin-converting enzyme 2 gene polymorphisms to essential hypertension and ischemic stroke].

OBJECTIVE: To study the relationship between angiotensin-converting enzyme 2 (ACE2) gene polymorphisms and the risk factor for essential hypertension (EH) with concurrent ischemic stroke in southern Chinese population. METHODS: The G9570A polymorphism in ACE2 gene were detected in 139 patients with EH and stroke using polymerase chain reaction-restriction fragment length polymorphism. Detailed clinical and biochemistrical data of the patients, including the pulse pressure, high sensitivity C-reactive protein (hsCRP), intima-media thickness (IMT), high-density lipoprotein cholesterol (HDL-C) and uric acid levels, were collected to study the relationship between ACE2 gene and the risk factor of EH and stroke. RESULTS: The levels of hsCRP (OR=1.022), uric acid (OR=1.224), IMT and pulse pressure was positively correlated to the incidence of EH and stroke. The pulse pressure, hsCRP, IMT, and HDL-C levels in male stroke patients carrying A allele was significantly higher than those in patients carrying G allele (P<0.05). In female stroke patients, the pulse pressure, hsCRP, IMT, and HDL-C levels were also significantly different with regard to the genotype of ACE2 gene (P<0.05). CONCLUSIONS: The patients with EH and ischemic stroke carrying the A/AA allele of ACE2 gene have higher risks than those carrying other allele, and can be also more vulnerable to stroke recurrence.

Heterologous expression and biochemical characterization of alpha-glucosidase from Aspergillus niger by Pichia pastroris.

The aglu of Aspergillus niger encodes the pro-protein of alpha-glucosidase, and the mature form of wild-type enzyme is a heterosubunit protein. In the present study, the cDNA of alpha-glucosidase was cloned and expressed in Pichia pastoris strain KM71. The activity of recombinant enzyme in a 3 L fermentor reached 2.07 U/mL after 96 h of induction. The recombinant alpha-glucosidase was able to produce oligoisomaltose. The molecular weight of the recombinant enzyme was estimated to be about 145 kDa by SDS-PAGE, and it reduced to 106 kDa after deglycosylation. The enzymatic activity of recombinant alpha-glucosidase was not significantly affected by a range of metal ions. The optimum temperature of the enzyme was 60 degrees C, and it was stable below 50 degrees C. The enzyme was active over the range of pH 3.0-7.0 with maximal activity at pH 4.5. Using pNPG as substrate, the K(m) and V(max) values were 0.446 mM and 43.48 U/mg, respectively. These studies provided the basis for the application of recombinant alpha-glucosidase in the industry of functional oligosaccharides.

[Construction of rat bowel transforming growth factor beta1 recombinant adenovirus vector].

OBJECTIVE: To construct a recombinant adenovirus vector which regulates the expression of rat transforming growth factor beta (TGF-beta1). METHODS: Total RNA was extracted from F344/N rat small intestine pre-treated with Con A to clone TGF-beta1. pTRE-shuttle vector was used as mediator to ligate TGF-beta1 gene and backbone of replication-incompetent adenoviral vector. The constructed recombinant adenovirus contained tetracycline-responsive element which could regulate the expression of inserted genes. After identification, the desired recombinant adenovirus was packaged in HEK 293 cells. Supernatant of high titer adenovirus was collected to detect the TGF-beta1 gene expression by green fluorescent protein(GFP). RESULTS: The constructed recombinant adenovirus was identified by restriction endonucleases cutting, sequencing, PCR and GFP examination. CONCLUSION: Rat TGF-beta1 recombinant adenovirus is established successfully, which provides material and evidence for further research of dendritic cell (DC) modified by TGF-beta1 to induce immune tolerance in rat heterotopic small bowel transplantation.

[Expression of mucosal addressin cell adhesion molecule-1 during small bowel graft rejection in rat].

OBJECTIVE: To investigate the expression of mucosal addressin cell adhesion molecule-1(MAdCAM-1) during small bowel graft rejection and the effects of MAdCAM-1 on the development of acute rejection. METHODS: Rat heterotopic small bowel transplantation (SBT) was performed in F344/N rats with syngeneic and allogeneic (BN-F344/N) grafts. Bowel and gut-associated lymphoid tissue(GALT) samples were collected from small bowel transplants on postoperative day(POD) 1, 3, 5 and 7. Histopathology assessment of the grafts was conducted to identify the rejection. MAdCAM-1 was detected by immunohistochemistry and Western blot. RESULTS: During acute rejection, MAdCAM-1 was highly-expressed on gut lamina propia and GALTs, particularly on vascular endothelial cells in the gut lamina propia. There were no change of MAdCAM-1 expression in syngeneic grafts from POD1 to POD7. In allogeneic grafts, MAdCAM-1 expression in mesenteric lymph nodes was down-regulated, while up-regulated on the vascular endothelial cells in the lamina propria during acute rejection. CONCLUSION: Alteration of MAdCAM-1 expression may be associated with the development of SBT graft rejection.

[Preparation and characterization of monoclonal antibodies against human RANTES molecule].

AIM: To prepare monoclonal antibodies against human RANTES molecule and identify the expression of RANTES in rat small intestine after small bowel transplantation. METHODS: Murine mAbs were prepared by B lymphocyte hybridoma technique. The expression of RANTES in rat small intestine after small bowel transplantation was detected by immunohistochemistry. RESULTS: Four hybridoma cell lines secreting monoclonal antibodies to human RANTES, FMU-RANTES 1, FMU-RANTES 2, FMU-RANTES 3 and FMU-RANTES 4, were established. The titers of a scetic mAbs reached to 1 x 10(-6) and the Ig subclass of FMU-RANTES 1, FMU-RANTES 3 and FMU-RANTES 4 was IgG1(kappa) and that of FMU-RANTES 2 was IgG2b(kappa). Among these mAbs, FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 3 could bind human RANTES protein in Western bolt. FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 4 could be used in immunohistochemistry staining. Rat RANTES molecule could be detected in the cyto plasm of epithelial cells in rat small intestine after small bowel transplantation. CONCLUSION: Four mAbs against RANTES molecule were prepared, which can provide a useful tool in research on the structure and function of RANTES molecule. High expression of RANTES may be involved in the rejection of allogeneic graft.