低氧诱导因子和角质细胞生长因子双基因重组腺病毒的构建及其在肺泡上皮细胞中的表达

目的：构建同时携带低氧诱导因子-1α（HIF-1α）和角质细胞生长因子（KGF）N腺病毒载体（pAdxsi-GFP-HIF-KGF）,观察其在防治肺损伤潜在的应用前景。方法：低氧处理A549细胞后提取总RNA并逆转录为eDNA作为模板,依据GeneBank公布的HIF-1α cDNA设计引物,并分别引入KpnI和BamHI酶切位点,PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上,构建重组质粒pShuttle-GFP—HIF。然后以质粒plRES2-EGFP-KGF为模板,用引入NheI和PmeI酶切位点的引物PCR扩增KGF基因并克隆到重组质粒pShuttle-GFP-HIF上,获得穿梭质粒重组质粒pShuttle—GFP-HIF—KGF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架栽体上构建携带HIF．10t和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF。检测重组腺病毒滴度后,转染人肺泡上皮细胞A549,检测目的基因的转染表达。结果：通过对构建质粒克隆进行测序及酶切,证实携带HIF—lot和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF构建成功,且构建的重组腺病毒纯度好、滴度高。用pAdxsi-GFP-HIF-KGF以100MOI转染A549细胞后24h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达,48h时荧光更强;转染48hELISA法检测培养上清中HIF-1蛋白表达水平为（56．36±4．53）ng／mL,KGF蛋白表达水平为（60．20±2．92）ng／mL。结论：成功构建了腺病毒栽体pAdxsi-GFP-HIF-KGF,其转染效率及目的基因的蛋白表达水平较高,具有潜在的进一步在肺损伤局部应用的前景,为后期制备可以同时发挥KGF、HIF-1作用的基因治疗药物打下基础,同时为高海拔地区应激性急性肺损伤的有效防治提供实验基础。     

RP1 Is a Phosphorylation Target of CK2 and Is Involved in Cell Adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser²³⁶ in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP²³⁶ show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser²³⁶ by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.     

Hypoxia Inducible Carbonic Anhydrase IX,Marker of Tumour: Hypoxia,Survival Pathway and Therapy Target

Over 50 genes are inducible by hypoxia, via hypoxia inducible factor 1a (HIF-1a). Carbonic anhydrase IX (CAIX) is one of the most inducible and most uniformly induced genes and because of its stability and membrane location provides a reliable histochemical marker of hypoxia. Recent studies have shown the importance of pH in cell death under hypoxia, thus mechanisms of pH regulation are likely to be vital pathways for survival. Carbonic anhydrases have a widespread role in normal tissues in regulating pH, with 14 isoforms described, so inhibition may have substantial normal tumour toxicity. Selective non-membrane permeable inhibitors are available and may synergise with chemotherapy agents more active in acid conditions. CAIX has a major role in regulating hydrogen ion (H+) flux and blockade of CAIX results in increased cell death under hypoxia, indicating that it is one mechanism of hypoxic adaptation. As it is commonly expressed in tumours with the worst prognosis it is a potential target for therapy.     

Characterization of aid1, a Novel Gene Involved in Fusobacterium nucleatum Interspecies Interactions

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.     

A Novel Role for the Hypoxia Inducible Transcription Factor HIF-1alpha: Critical Regulation of Inflammatory Cell Function

No abstract available.     

The Histone Deacetylase Inhibitor,Vorinostat, Represses Hypoxia Inducible Factor 1 Alpha Expression through Translational Inhibition

Hypoxia inducible factor 1α (HIF-1α) is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi) block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA) and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor - eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.     

氯化钴对H9c2 心肌细胞新基因Mipu1 表达的影响

目的:研究氯化钴(CoCl2)对大鼠胚胎心脏来源的H9c2心肌细胞中新基因Mipu1表达的影响。方法:利用不同浓度的CoCl2(0、100、200、300、400、500μmol/L)处理H9c2细胞9h,及200μmol/L CoCl2处理H9c2细胞不同的时间(0、6、9、12、24h)后,用RT-PCR和Western Blot分别观察H9c2细胞Mipu1 mRNA和蛋白的表达情况。结果:CoCl2可以诱导H9c2细胞中Mipu1 mRNA和蛋白表达升高,200μM CoCl2处理组的Mipu1的表达水平高于100μM CoCl2处理组,但是更高浓度的CoCl2(>200μM)不能使Mipu1的表达进一步升高。随着CoCl2作用时间的延长,Mipu1的表达逐步升高,在12h达到高峰,但是在24h后下降。结论:CoCl2能够促进H9c2细胞新基因Mipu1的表达,并且具有一定的剂量和时间依赖性。