半巢式甲基化特异性聚合酶链反应在胃癌p16基因甲基化检测中的应用

目的　改进甲基化特异性聚合酶链反应 (MSP) ,采用半巢式MSP检测胃癌组织p16基因启动子区CpG岛的甲基化状态。方法　用亚硫酸氢盐修饰被测DNA后 ,采用半巢式MSP(引物分别为MS ,M1A和MS ,M2A) ,分析了 6 0例胃癌组织及相应正常组织中p16基因的甲基化状态。结果　单独用MSP ,胃癌组织中p16基因甲基化的发生率为 80 %。采用半巢式MSP ,甲基化发生率为86 7% ,比单独采用MSP提高了几个百分点 ,并提示胃癌病例的p16基因启动子区存在甲基化发生的不同模式。结论　半巢式MSP能够有效地提高灵敏度和减少假阳性。同时 ,免疫组化的结果也说明了p16基因异常甲基化与胃癌组织P16蛋白的表达密切相关。     

Deletion of p16 and p15 genes In schistosomiasis-associated bladder cancer (SABC)

Alterations of p16 and p15 genes have been reported in cancer cell lines and in certain malignant neoplasm. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators at G₁-S checkpoint. One hundred and sixty eight tumor tissue, 20 schistosomal tissue, and 50 normal tissue samples were examined. The status of p16 and p15 genes in these tissues was determined by the polymerase chain reaction and by sequencing the DNA fragments produced during PCR. In addition, the expression of p16 and p15 proteins was examined by Western blot analysis. p16 and p15 genes were detected in all normal and schistosomal tissues. Deletion of both p16 and p15 genes was observed in 72 and 36 bladder tumors, respectively. Twenty eight of the 72 cases that exhibited p16 deletions also displayed deletions of p15. Only eight cases showed loss of the p15 gene while retaining p16 gene, and p16 deletion with apparently intact p15 gene was identified in 44 cases. The present analysis also reveals that deletion in the two genes are associated with low-stage, low grade bladder cancer, schistosomiasis-associated bladder cancer (SABC) and squamous cell carcinoma type (SCC). No point mutations were identified in either gene. The expression of p16 and p15 proteins was undetectable in 75 and 38 bladder tumors, respectively, by Western blot analysis. Alteration of the p16 and p15 genes appears to be an early event in bladder cancer which occurs more frequently in SABC and SCC, and may play an important role in the development of schistosomal bladder cancer.     

Variation of gene expression profile linked to p27 Ser phosphorylation status in MCF-7 cell line

Ser¹⁰ was the major phosphorylation site in p27^Kip1 (here after referred to as p27), accounting for 70% of the total phosphorylation of this protein, due to cytoplasmic shuttling. To further elucidate the mechanism for Ser¹⁰-mediated dysfunction of p27 during breast cancer progression. Affymetrix Human Genome U133A Array was used to identify differentially regulated genes between MCF-7 breast cancer cell lines stably-transfected with wild type and Ser¹⁰ mutant (substitution of Ser¹⁰ with Ala, S10A), alternatively. Then to confirm by RT-PCR then western blot partly. Microarray analysis showing that S10A, then abrogation of Ser¹⁰ phosphorylation, result in important changes in a large number of genes involved in the control of cell cycle, cell differentiation, metabolism, immune response and signal transduction. S10A induced cell cycle G₀ phase arrest by FACS method. And cell growth inhibition and abrogation of cytoplasmic translation. Our data indicate that abrogation of phosphorylation of Ser¹⁰ resulted in significant changes in gene expression profiles which mediate cell cycle redistribution, sub-cellular localization.     

Influence of UGT1A1 gene methylation level in colorectal cancer cells on the sensitivity of the chemotherapy drug CPT-11

Objective

To study the influence of the methylation level of UGT1A1 gene related to CPT-11 metabolic enzymes in colorectal cancer cells on the sensitivity of chemotherapy drugs.

Methods

Test the changes in sensitivity of seven colorectal cancer cell strains that have been/not been subject to DAC treatment to CPT-11, analyze its correlation with CES2, UGT1A1 and GUSB mRNA expression according to IC₅₀; screen the effective interference sequence of UGT1A1 siRNA, test the changes in cytotoxicity of CPT-11 after UGT1A1 siRNA is transfected, select RK0 cells and make them transfected with the chemosynthetic UGT1A1 siRNA after their UGT1A1 expression is restored with or without demethylation treatment.

Results

The sensitivity of different colorectal cancer cell strains to CPT-11 showed difference (P < 0.05), UGT1A1 expression in colorectal cell lines had a negative correlation with the IC₅₀ (r = 0.790648, P < 0.05), the interference efficiency of the screened UGT1A1 siRNA was up to 78%. The IC₅₀ value of siRNA decreased by nearly one time after transfected with HT-29 (P < 0.01); which of methylated RK0 cells of UGT1A1 gene increased instead after the demethylation treatment. However, the IC₅₀ value of the demethylation treatment group increased compared with the non-demethylation treatment group after UGT1A1 siRNA was transfected.

Conclusions

The cytotoxicity of CPT-11 to colorectal cancer cells has a negative correlation with UGT1A1 expression, and positive correlation with CES2 and GUSB. The specific silencing UGT1A1 gene of siRNA could significantly increase the sensitivity of CPT-11 to the chemotherapy of colorectal cancer cells. UGT1A1 methylation was an important factor affecting the chemosensitivity of CPT-11.     

HPV-DNA检测联合p16～（INK4A）蛋白表达对于子宫颈腺癌的诊断意义研究

目的：探讨HPV-DNA检测联合P16～（INK 4A ）蛋白表达对子宫颈腺癌的诊断意义。方法选取2013年5月至2014年6月在本院接受治疗的宫颈腺癌变患者125例,其中CINⅠ患者40例,CINⅡ患者30例,CINⅢ患者25例,浸润性腺癌患者30例,同时选取25例正常宫颈组织,采用免疫组织化学方法检测患者组织中的P16INK 4A ,同时采用第二代杂交捕获法对患者体内的13种HPV-DNA进行检测。结果检测结果表明,宫颈腺癌病变中CINⅠ、CINⅡ、CINⅢ组织以及浸润性腺癌组织中HPV-DNA感染的阳性率以及P16INK4A蛋白表达的阳性率都明显的高于正常宫颈组织,进行比较差异显著（P＜0．05）;宫颈组织中 HPV-DNA感染的阳性率与P16INK 4A蛋白表达的阳性率呈正相关关系。结论 HPV-DNA检测联合P16～（INK 4A）蛋白表达对子宫颈腺癌的诊断,有利于提高自宫颈腺癌以及CIN的阳性预测值。     

A novel bla(CTX-M-14) gene-harboring complex class 1 integron with an In4-like backbone structure from a clinical isolate of Escherichia coli

Escherichia coli BDE0502 contained 3 beta-lactamase genes including bla(DHA-1), bla(SHV-12), and bla(CTX-M-14). The bla(CTX-M-14) gene was found on a complex class 1 integron with an In4-like backbone structure. The bla(CTX-M-14) gene was proceeded by a partial copy of ISEcp1 in addition to the complete copy of ISCR1 element.     

Cloning and sequencing of the class A beta-lactamase gene (blaTEM-15) located on a chromosomal Tn801 transposon

Escherichia coli CA0210 was identified in a stool culture of a 03-month-neonate in Tunisia. This strain was resistant to beta-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefpirome, ceftazidime, and cefotaxime, but it remained susceptible to imipenem and cefoxitin. The beta-lactam-hydrolyzing beta-lactamase gene of E. coli CA0210 and the upstream and downstream regions were cloned, sequenced, and expressed in E. coli DH5alpha. These resistances were carried by a 1080-bp chromosomal gene that encoded a beta-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that the corresponding blaTEM-15 gene was part of a chromosomally located Tn801 transposon.     

Partial inefficiency of T cell receptors γ/δ composed of a heavy (55-kD) γ chain to mediate cell activation upon binding to specific monoclonal antibodies

Summary We analyzed CD8⁺ T cell receptor (TCR) γ/δ⁺ (δ-TCS-1 reactive) cell clones expressing the 55-kD γ chain for their susceptibility to triggering by monoclonal antibodies (mAbs) specific for TCR or CD3 molecules. Clones were derived by limiting dilution from CD3⁺, WT31⁻ FACS-purified peripheral blood populations or CD4⁻CD8⁻ thymocytes (a fraction of the latter cells expressingde novo CD8 surface antigen upon culture in IL-2). Clones were screened according to their reactivity with both anti-CD8 and δ-TCS-1 mAbs. Analysis of CD3-associated molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions which preserve the CD3/TCR association (1% digitonin) showed a predominant 55–60-kD molecule both under reducing and non-reducing conditions. All clones expressing the δ-TCS-1⁺ CD8⁺ surface phenotype derived from either thymus or peripheral blood lysed the Fcγ receptor-bearing P815 target cells in the presence of anti-CD3 mAb. On the other hand, δ-TCS-1 mAb was poorly efficient in triggering the lytic machinery of these clones, while it induced target cell lysis by δ-TCS-1⁺ CD8⁻ clones. This work was supported by grants from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy,Progetto Finalizzato ‘Oncologia’ to M. C. M. and A. M., and from theAssociazione Italiana per la Ricerca sul Cancro (AIRC).     

Extended characterisation of the serotonin 2A (5-HT2A) receptor-selective PET radiotracer 11C-MDL100907 in humans: quantitative analysis, test-retest reproducibility, and vulnerability to endogenous 5-HT tone

Introduction

Scanning properties and analytic methodology of the 5-HT_2A receptor-selective positron emission tomography (PET) tracer ¹¹C-MDL100907 have been partially characterised in previous reports. We present an extended characterisation in healthy human subjects.

Methods

64 ¹¹C-MDL100907 PET scans with metabolite-corrected arterial input function were performed in 39 healthy adults (18-55 years). 12 subjects were scanned twice (duration 150 min) to provide data on plasma analysis, model order estimation, and stability and test-retest characteristics of outcome measures. All other scans were 90 min duration. 3 subjects completed scanning at baseline and following 5-HT_2A receptor antagonist medication (risperidone or ciproheptadine) to provide definitive data on the suitability of the cerebellum as reference region. 10 subjects were scanned under reduced 5-HT and control conditions using rapid tryptophan depletion to investigate vulnerability to competition with endogenous 5-HT. 13 subjects were scanned as controls in clinical protocols. Pooled data were used to analyse the relationship between tracer injected mass and receptor occupancy, and age-related decline in 5-HT_2A receptors.

Results

Optimum analytic method was a 2-tissue compartment model with arterial input function. However, basis function implementation of SRTM may be suitable for measuring between-group differences non-invasively and warrants further investigation. Scan duration of 90 min achieved stable outcome measures in all cortical regions except orbitofrontal which required 120 min. Binding potential (BP_P and BP_ND) test-retest variability was very good (7-11%) in neocortical regions other than orbitofrontal, and moderately good (14-20%) in orbitofrontal cortex and medial temporal lobe. Saturation occupancy of 5-HT_2A receptors by risperidone validates the use of the cerebellum as a region devoid of specific binding for the purposes of PET. We advocate a mass limit of 4.6 μg to remain below 5% receptor occupancy. ¹¹C-MDL100907 specific binding is not vulnerable to competition with endogenous 5-HT in humans. Paradoxical decreases in BP_ND were found in right prefrontal cortex following reduced 5-HT, possibly representing receptor internalisation. Mean age-related decline in brain 5-HT_2A receptors was 14.0 ± 5.0% per decade, and higher in prefrontal regions.

Conclusions

Our data confirm and extend support for ¹¹C-MDL100907 as a PET tracer with very favourable properties for quantifying 5-HT_2A receptors in the human brain.     

Past, present and future of A(2A) adenosine receptor antagonists in the therapy of Parkinson's disease

Several selective antagonists for adenosine A_2A receptors (A_2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson's disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D₂ and adenosine A_2A receptors in the basal ganglia. At present it is believed that A_2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson's patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A_2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized antiparkinsonian drug therapy, namely the existence of (hetero)dimers/oligomers of G protein-coupled receptors, a topic that is currently the focus of intense debate within the scientific community. Dopamine D₂ receptors (D₂Rs) expressed in the striatum are known to form heteromers with A_2A adenosine receptors. Thus, the development of heteromer-specific A_2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs.     

The A640G polymorphism in the NAD(P)H oxidase p22phox gene (CYBA) is associated with risk reduction of coronary heart disease: A meta-analysis

Background

Recently, studies have focused on the association between the p22phox gene A640G polymorphism and coronary heart disease (CHD). However, the results are inconsistent. In this study, we aimed to further evaluate this association by using meta-analysis.

Methods

The PubMed, Embase, CBM, CNKI, WanFang and Chongqing VIP databases were searched for relevant articles. Hardy–Weinberg equilibrium (HWE) of the distribution of genotypes was tested using Pearson's chi-squared test. Odds ratios (ORs) with the corresponding 95% confidence intervals (CIs) were used to assess the strength of the association; Cochran's Q test and the I² statistic were used to evaluate heterogeneity. The random effects model and the fixed effects model were used according to heterogeneity; Begg's test and Egger's test were used to analyze publication bias. Sensitivity analysis was carried out to guarantee the stability of the results. Cumulative analysis was used to evaluate tendencies in the pooled OR.

Results

A total of eight articles including 3904 CHD cases and 3498 controls were included. A significant association between the A640G polymorphism and CHD was observed in codominant model 2 (AG versus AA: OR = 0.86, 95% CI: 0.77–0.96). In the subgroup analysis, a significant association was observed between the A640G polymorphism and CHD in Caucasians, and in PB (population-based), non-PB, HWE (studies followed HWE) and non-HWE studies.

Conclusions

Our results reveal that the A640G polymorphism may play a protective role in CHD.     

The biology of Nociceptin/Orphanin FQ (N/OFQ) related to obesity,stress, anxiety,mood, and drug dependence

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995. The generation of specific agonists, antagonists and receptor deficient mice and rats has enabled progress in elucidating the biological functions of N/OFQ. Additionally, radio-imaging technologies have been advanced for investigation of this system in animals and humans. Together with traditional neurobehavioral techniques, these tools have been utilized to identify the biological significance of the N/OFQ system and its interacting partners. The present review focuses on the role of N/OFQ in the regulation of feeding, body weight homeostasis, stress, the stress-related psychiatric disorders of depression and anxiety, and in drug and alcohol dependence. Critical evaluation of the current scientific preclinical literature suggests that small molecule modulators of nociceptin opioid peptide receptors (NOP) might be useful in the treatment of diseases related to these biological functions. In particular, the literature data suggest that antagonism of NOP receptors will produce anti-obesity and antidepressant activities in humans. However, there are also contradictory data discussed. The current literature on the role of N/OFQ in anxiety and addiction, on the other hand points primarily to a role of agonist modulation being potentially therapeutic. Some drug-like molecules that function either as agonists or antagonists of NOP receptors have been optimized for human clinical study to test some of these hypotheses. The discovery of PET ligands for NOP receptors, combined with the pharmacological tools and burgeoning preclinical data set discussed here bodes well for a rapid advancement of clinical understanding and potential therapeutic benefit.