Hypoxia-induced nucleophosmin protects cell death through inhibition of p53

Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.     

人X染色体间期细胞遗传学研究

应用人X染色体α卫星DNA探针进行X染色体正常或异常个体的外周血淋巴细胞染色体和间期核的原位杂交,在R显带的中期分裂相上,绝大部分杂交颂粒位于X染色体着丝粒区(p11→q11);在间期核内则显现与X染色体数相一致的银颗粒簇,其中相当部分位于核边缘区。实验结果表明,用原位杂交来检测X染色体数目,比记数Barr小体的方法可靠。本文还就α卫星DNA探针在间期细胞遗传学方面广泛的应用做了讨论。     

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.     

斜纹夜蛾核多角体病毒Spit119基因在大肠杆菌中的表达及抗体制备

用PCR的方法扩增得到斜纹夜蛾核多角体病毒(Spodoptera litura nucleopolyhedrovirus,SpltMNPV)ORF119全长基因(Splt119),将其克隆至5'端有6 × His编码序列的原核表达栽体pQE30上,转化大肠杆菌M15[pREP4],在1 mmol/LIPTG诱导下超量表达了一个分子量约28 kD的融合蛋白.经聚丙烯酰胺凝胶电泳纯化,免疫新西兰大白兔制备了特异的抗Splt119抗体.Western blotting免疫印迹分析表明,该抗体适合用作Splt119蛋白功能的进一步研究.     

薇甘菊精油化学成分及其对小菜蛾的驱避及产卵抑制作用(英文)

用四臂嗅觉仪测定了小菜蛾对薇甘菊挥发油、芋烯、α-松油烯、里哪醇、β-石竹烯、马鞭草烯酮6种挥发物嗅觉反应,在网室,比较了挥发油及5种挥发物对小菜蛾雌虫产卵的驱避效果。结果表明:薇甘菊挥发油不仅对小菜蛾成虫有明显的驱避作用,而且能显著地减少其成虫在寄主植物上的产卵量。薇甘菊挥发油所含的5种挥发性化合物中芋烯、α-松油烯和里哪醇表现出较强的驱避效果(流量为100-180mL/min)和产卵抑制作用(剂量为10-20μL/株),而小菜蛾对马鞭草烯酮和β-石竹烯的嗅觉反应以及小菜蛾在这两种化合物处理的寄主植物上的产卵量与对照相比无显著差异。     

Identification of Vibrio harveyi using PCR amplification of the toxR gene

AIMS: The aim of this study was to develop an effective method for the identification of Vibrio harveyi based on using the toxR gene as a taxonomic marker. METHODS AND RESULTS: Primers for the toxR gene were designed for specificity to V. harveyi, and incorporated in a polymerase chain reaction (PCR). The results of the PCR, which took <5 h from DNA extraction to amplification, revealed positive amplification of the toxR gene fragment in 20 V. harveyi isolates including type strains, whereas DNA from 23 other Vibrionaceae type strains and 13 Vibrio parahaemolyticus strains were negative. The detection limit of the PCR was 4.0 x 10(3) cells ml(-1). In addition, the technique enabled the recognition of V. harveyi from diseased fish. CONCLUSIONS: The PCR was specific and sensitive, enabling the identification of V. harveyi within 5 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR allowed the rapid and sensitive detection of V. harveyi.     

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII.     

Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B₁ PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.