NF-kappaB and COX-2 during oral tumorigenesis and in assessment of minimal residual disease in surgical margins

Oral cancer is a major health problem in many parts of the world including India. The molecular mechanisms involved in oral tumorigenesis are not completely understood. Although surgery continues to be the most common treatment modality for this cancer, survival rates of oral cancer patients have still not significantly improved over the last few decades. Classical diagnostic methods are still not sensitive enough in detecting completeness of surgery and assessing minimal residual disease. This study investigated the role of NF-kappaB and COX-2 both in oral cancer progression and assessment of minimal residual disease. Expression of NF-kappaB proteins and its inhibitory protein IkappaB-alpha was evaluated using immunohistochemistry, ELISA and EMSA, while RT-PCR was used to detect COX-2 expression. Cytoplasmic expression as well as nuclear translocation of NF-kappaB proteins increased with histological progression of oral cancer (from normal to leukoplakia to cancer). A similar pattern of expression was observed for COX-2 also. NF-kappaB proteins, both cytoplasmic and nuclear, had a significant negative correlation from tumor to surgical margin to extra margin; COX-2 paralleled the expression of NF-kappaB proteins. Our results thus point to NF-kappaB and COX-2 as participants in oral tumor progression and also to the validation of these two molecular markers in assessing minimal residual disease.     

急性心肌缺氧对乳鼠心肌细胞脑钠尿肽表达的影响及其作用机制

目的: 观察急性缺氧损伤对乳鼠心肌细胞脑钠尿肽(BNP)表达水平的影响并探讨其可能作用机制。方法: 原代乳鼠心肌细胞缺氧、无糖、无血清培养以模拟心肌缺血损伤,利用CCK-8法测细胞存活率、ELISA法测白细胞介素-6(IL-6)和BNP的表达;以IL-6直接干预体外培养的心肌细胞, 采用RT-PCR、Western blotting和ELISA方法观察BNP、转化生长因子β₁(TGF-β₁)、Smad2 mRNA的转录和蛋白的表达。结果: 缺氧显著上调IL-6和BNP的表达,且两者呈线性正相关;IL-6直接干预可剂量和时间依赖性地上调心肌细胞BNP的mRNA和蛋白表达水平,同时TGF-β₁和Smad2的表达水平亦增加;而针对TGF-β₁的中和抗体能够部分地抑制由IL-6引起的BNP表达增加。结论: 急性心肌缺氧可直接上调BNP的表达水平,这种调节效应与TGF-β₁/Smad2信号通路上调有关。     

Effect of annexin A2 on hepatopulmonary syndrome rat serum-induced proliferation of pulmonary arterial smooth muscle cells

Hepatopulmonary syndrome (HPS) is characterizes by an arterial oxygenation defect induced by intrapulmonary vasodilation that increase morbidity and mortality. However, the underlying mechanisms on HPS-associated pulmonary vascular remodeling remains undefined. In this study, we found that HPS rat serum, drawn from common bile duct ligation (CBDL) rats, mediated the overexpression of ANXA2 and the proliferation of PASMCs. And small interfering RNA (siRNA) that target rat ANXA2 led to significant downregulation of ANXA2, which resulted in the decreased proliferation of PASMCs. Subsequently, we further examined the role of ANXA2 siRNA in the regulation of pro-proliferative signaling such as that mediated by ERK1/2 and NF-κB, and found the attenuation of HPS-associated activation of the signaling pathway. Thus, the fact highlighted the crucial role of ANXA2 in HPS-associated PASMC proliferation, and suggested a potential therapeutic effect on HPS-associated pulmonary vascular remodeling.     

转化生长因子β1对乳鼠心肌细胞转化生长因子结合蛋白2表达的影响

 目的：探讨转化生长因子β1（TGF-β1）在诱导心肌细胞表达转化生长因子结合蛋白2（LTBP2）中的作用及信号传导通路。 方法：培养乳鼠心肌细胞;实时定量聚合酶链式反应（Real-time PCR）、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制。 结果：LTBP2基因表达随着TGF-β1浓度增加（0、2、5、10 ng/mL）而明显升高,在5 ng/mL时刺激最强（P < 0.05）;5 ng/mL的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势（P < 0.05或P<0.01）;免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达。信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达。 结论：TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达。     

匹罗卡品致痫大鼠脑内NF-κB及COX-2的表达

为探讨癫痫大鼠脑内核转录因子NF-κB及炎性因子环氧化酶-2(COX-2)的表达,本研究采用匹罗卡品(30mg/kg)诱导大鼠癫痫发作后,通过免疫组织化学和Westernblot方法,对癫痫大鼠脑内,主要是海马NF-κB和COX-2的表达进行了观察。结果显示:匹罗卡品诱导大鼠癫痫发作后,大鼠脑内尤其是海马神经元的胞浆和胞核内NF-κBp65免疫反应阳性增强,核移位增多,蛋白产物表达增多;COX-2免疫反应阳性明显增强,蛋白产物表达增多。以上结果提示,癫痫发作后可引起核转录因子NF-κB的活化及炎性因子COX-2的高表达。     

Effects of ghrelin on protein expression of antioxidative enzymes and iNOS in the rat liver

Introduction

We investigated the effects of ghrelin on protein expression of the liver antioxidant enzymes superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), nuclear factor κB (NFκB) and inducible nitric oxide synthase (iNOS). Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK1/2) and protein kinase B (Akt) are involved in ghrelin-regulated liver antioxidant enzymes and iNOS protein expression.

Material and methods

Male Wistar rats were treated with ghrelin (0.3 nmol/5 µl) injected into the lateral cerebral ventricle every 24 h for 5 days, and 2 h after the last treatment the animals were sacrificed and the liver excised. The Western blot method was used to determine expression of antioxidant enzymes, iNOS, phosphorylation of Akt, ERK1/2 and nuclear factor κB (NFκB) subunits 50 and 65.

Results

There was significantly higher protein expression of CuZnSOD (p < 0.001), MnSOD (p < 0.001), CAT (p < 0.001), GPx, (p < 0.001), and GR (p < 0.01) in the liver isolated from ghrelin-treated animals compared with control animals. In contrast, ghrelin significantly (p < 0.01) reduced protein expression of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 was significantly (p < 0.001 for p65; p < 0.05 for p50) reduced by ghrelin when compared with controls. Phosphorylation of ERK1/2 and of Akt was significantly higher in ghrelin-treated than in control animals (p < 0.05 for ERK1/2; p < 0.01 for Akt).

Conclusions

The results show that activation of Akt and ERK1/2 is involved in ghrelin-mediated regulation of protein expression of antioxidant enzymes and iNOS in the rat liver.     

Regulation of Drosophila-virus interaction

Drosophila melanogaster is a useful model system for deciphering mammalian biological processes including development, innate immunity and cancer. Most genetic studies conducted in Drosophila have focused on the immune response against microbial infection and the results obtained have been extrapolated to other organisms. During the last decade the issue of the antiviral response attracted a great deal of interest. In this review we highlight recent discoveries in the role of RNA interference pathway in antiviral response in Drosophila with a focus on the role of miRNAs as both host defense elements and helpers of viral replication.     

Latent membrane protein 2A inhibits expression level of Smad2 through regulating miR-155-5p in EBV-associated gastric cancer cell lines

Epstein-Barr virus (EBV) infection is one of the causes of gastric cancer (GC). Besides, previous studies have demonstrated that EBV-encoded latent membrane protein 2A (LMP2A) influences the pathogenesis of EBV-associated gastric cancer (EBVaGC) through regulating several key pathways. In this study, the expression level of Smad2 was observed, which was reduced in EBVaGC cell lines, especially in the presence of LMP2A. Meanwhile, we found that LMP2A promoted the expression of miR-155-5p by activated nuclear factor-κB (NF-κB) signaling. After being treated with NF-κB inhibitor (BAY 11-7082), miR-155-5p sharply decreased. Western blot analysis proved that the overexpression of miR-155-5p could inhibit Smad2. Functional studies showed that the role of miR-155-5p might lead to good prognosis in EBV-positive GC through promoting cell apoptosis and cell cycle arrest, as well as inhibiting tumor proliferation. In addition, p-Smad2 protein was also reduced or induced by overexpression or knockdown, respectively, of miR-155-5p. Immunofluorescence analysis further indicated that LMP2A prevented p-Smad2 from transferring to the nucleus, which played a crucial role in transforming growth factor-β (TGF-β) signaling. In summary, our findings confirmed the relationship between LMP2A and Smad2 and provided a potential regulation of the TGF-β pathway in EBVaGC.     

Differential expression of toll-like receptor signaling cascades in LPS-tolerant human peripheral blood mononuclear cells

Pre-exposure to low doses of LPS induces resistance to a lethal challenge, a phenomenon known as endotoxin tolerance. In this study, tolerance was induced in human PBMC by culturing cells with 1 ng/mL LPS for 48 h. Cells were subsequently challenged with 100 ng/mL LPS for 2, 6 and 24 h, and the expression of 84 genes encoding proteins involved in the TLR signaling pathway was evaluated at each time point by PCR array. LPS pretreatment did not modulate the expression of TLR4 and CD14 on the surface of monocytes. A gene was defined as tolerized when LPS pretreatment reversed the effect of LPS challenge on the expression of the gene or as non-tolerized when LPS pretreatment did not reverse the effects of LPS challenge. We observed impaired signal transduction through the NF-κB, JNK, ERK and TRIF pathways, whereas expression of p38 pathway-related genes was preserved in LPS-tolerant cells. These results show a distinct regulation of the TLR pathway cascades during tolerance; this may account for the differential gene expression of some inflammatory mediators, such as up-regulation of IL-10 and COX2 as well as down-regulation of TNF-α and IL-12. Depending on the effect of LPS-induced gene up-regulation or down-regulation, tolerance, as a reversion of such LPS effects, may result in repression or induction of gene expression.     

转化生长因子β1/Smad信号通路对大鼠肾系膜细胞基质金属蛋白酶2及其组织抑制因子2表达的影响

目的　探讨转化生长因子 β1(TGF β1) /Smad信号通路对大鼠肾系膜细胞 (MsC)基质金属蛋白酶 2 (MMP 2 )及其组织抑制因子 2 (TIMP 2 )蛋白表达和酶活性的影响。方法　采用脂质体介导法 ,分别将Smad 2 3 7基因瞬时转染体外培养的大鼠MsC ,并用免疫荧光 逆转录 聚合酶链反应 (RT PCR)和Western印迹法检测其转染成功与否 ;再分别用Western印迹 酶谱法或反式酶谱法 ,观察转基因MsC及其在TGF β1作用下MMP 2和TIMP 2蛋白表达和酶活性的改变。 结果　转染Smad 2基因的MsC ,MMP 2和TIMP 2蛋白表达和酶活性均略有升高 ,蛋白表达分别增强 1 4倍和 1 1倍 ;酶活性分别提高 1 4倍和 1 3倍 ,经TGF β1作用后有明显升高 ,蛋白表达分别增加 2 8倍和 3 0倍 (P <0 0 1) ;酶活性分别提高 3 0倍和 1 7倍 (P <0 0 1) ;转染Smad 3基因的MsC ,TIMP 2蛋白表达和酶活性略有升高 ,蛋白表达增强 1 2倍 ,酶活性提高 1 2倍 ,TGF β1作用后升高更为明显 ,蛋白表达增强 2 9倍 ,酶活性提高 1 8倍 ,而MMP 2蛋白表达和酶活性均无明显变化 ;转染Smad 7基因的MsC ,MMP 2和TIMP 2蛋白表达和酶活性均略有下降 ,蛋白表达均降低 1 2倍 ,酶活性均下降 1 2倍 ,在TGF β1作用下更为明显 ,蛋白表达分别     

Mechanisms of genotype-phenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency

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Blaschko line analogies in the central nervous system: A hypothesis

In X-chromosome-linked skin disorders the pattern of involvement follows Blaschko lines. Patterns of changes analogous to cutaneous Blaschko lines in different X-linked diseases existed in other organs. There is no commonly accepted analogy to Blaschko lines in the central nervous system (CNS). The objective of this study was to consider a hypothesis of the existence of Blaschko lines in the CNS in the example of incontinentia pigmenti (IP). Articles were analyzed in which brain imaging methods were used in IP patients with CNS anomalies. In IP patients with neurological signs brain lesions usually were localized and extended radially. Affected areas did not correspond to territories vascularized by any determined artery. Radially distributed brain lesions morphologically match the radial unit model of cortical development. It can be proposed that in IP in CNS Blaschko line analogies, similar to those in the skin, represent the trace of development of the clone of neurons arising from the cell marked with IKBKG mutation. The hypothesis of the existence of Blaschko line analogies in CNS is supported by radially distributed CNS image findings in IP, the radial unit model of CNS development, and the common embryonic origin of skin, CNS, and eyes.     

急性肺损伤大鼠肺组织β和β2受体及β2受体mRNA表达的变化

目的：观察急性肺损伤（ＡＬＩ）大鼠肺组织β、β２受体和β２受体ｍＲＮＡ含量的变化,探讨上述变化与ＡＬＩ的关系及β２受体变化可能的分子机制。方法：静注内毒素复制大鼠ＡＬＩ模型,肺组织β、β２受体及β２受体ｍＲＮＡ含量,分别用放射性配基结合分析法和斑点杂交技术测定。结果：静注内毒素后１、４、６ｈ,大鼠肺组织β和β２受体的最大结合容量（Ｂｍａｘ）均明显低于正常对照组（Ｐ＜０．０１）,尤以β２受体为甚;静注内毒素后４及６小时,大鼠肺组织β２受体ｍＲＮＡ含量分别下降至正常对照组的７６４％±１９６％（Ｐ＜０．０１）和５２８％±２０４％（Ｐ＜０．０１）。结论：肺组织β受体数目的减少在ＡＬＩ发生发展中可能起一定的作用,β２受体数目的减少可能与ＡＬＩ关系更密切;β２受体ｍＲＮＡ含量的减少可能是ＡＬＩ后一定阶段肺组织β２受体数目减少的原因之一。