Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.     

灰金合欢根瘤类菌体繁殖及其宿主细胞的超微结构

根瘤细胞早期发育阶段,以宿主细胞器和根瘤菌转化类菌体的数量增多为特征。随后类菌体增殖到填满宿主细胞内的大部分区域。各个类菌体周膜内含有1至几个类菌体。晚期共生发育阶段,类菌体细胞结构和宿主细胞器数量发生了变化。文中还讨论了根瘤的共生固氮作用。     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Proteomic identification of the cerebral cavernous malformation signaling complex

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.     

Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G₁ phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB.     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

Biological Control of Carrot Black Rot

Diseased carrot seeds were treated with selected micro-organisms isolated from soils, carrot seeds and tap roots. The effects of those antagonists on the control of Alternaria radicina were evaluated by growing-on tests on water agar, filter paper, vermiculite and in a potting medium (BVB no. 4). The germination percentage, emergence percentage and the disease severity of those carrot seeds treated with Burkholderia (Pseudomonas) cepacia no.229 were significantly (P=0.05) differed from the non-treated seeds and the seed treated with other antagonists. The effects of B. cepacia no.229 in promoting seed emergence and controlling disease were as good as those seeds treated with iprodione (100 p.p.m.). Black rot lesions on carrot tap roots were significantly reduced (P=0.05) in size when roots were treated with B. cepacia no 229 or Bacillus amyloliquefaciens no. 224 compared to the nontreated roots. Also, B. cepacia no. 229 significantly (P=0.05) reduced black rot on the foliage of carrot compared to check.     

小RNA病毒和小DNA病毒的三维结构模型

本文综述了小RNA病毒和小DNA病毒的主要结构特征,绘制出了它们的三维结构模型,从中找出了两者间的共同点和差异,为进一步研究两种病毒提供了依据。     

MicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy

Autophagy is activated in cancer cells during chemotherapy and often contributes to tumor chemotherapy resistance. In this study, we characterized the role of microRNA-30a (miR-30a) in the coordination of cancer cell apoptosis and autophagy, which determines the sensitivity of cancer cells to chemotherapy. First, the autophagy activity in cancer cells increased after cis-dichloro-diamine platinum (cis-DDP) or Taxol treatment, as indicated by the enhanced expression of beclin 1, a key regulator of autophagy, and increased number of LC3-positive autophagosomes. Second, miRNA screening using a TaqMan probe-based quantitative RT-PCR assay identified that miR-30a, a miRNA that targets beclin 1, was significantly reduced in tumor cells by cis-DDP treatment. Forced expression of miR-30a significantly reduced beclin 1 and the autophagy activity of tumor cells induced by cis-DDP. Third, the blockade of tumor cell autophagy activity by miR-30a expression or 3-methyladenine significantly increased tumor cell apoptosis induced by cis-DDP treatment. Finally, an in vivo tumor implantation mouse model clearly showed that elevation of miR-30a in implanted tumor cells by administration of the recombinant lentivirus expressing miR-30a strongly enhanced cis-DDP-induced apoptosis of tumor cells. In conclusion, our results demonstrate for the first time that miR-30a can sensitize tumor cells to cis-DDP via reducing beclin 1-mediated autophagy and that increasing miR-30a level in tumor cells represents a novel approach to enhance the efficacy of chemotherapy during cancer treatment.     

碱茅(puccinellia tenuifolra)Put-Cu/Zn-SOD基因的克隆及在酵母中的表达

从碱茅根cDNA文库分离得到Put-Cu/Zn-SOD全长cDNA,其序列全长为2 700 bp,该基因的开放读码框为长615 bp,所编码的蛋白由204个氨基酸组成,其预测分子量约为20.6 kD、理论等电点约为5.63.Put-Cu/Zn-SOD氨基酸序列与水稻Cu/Zn-SOD序列有较高的同源性为87%.将Put-Cu/Zn-SOD基因构建到酵母表达载体pYES2,并转化至酵母(INVSc1).对pYES2-Put-Cu/Zn-SOD转化酵母进行盐碱、氧化胁迫实验.结果表明:重组酵母的抗盐碱、氧化能力明显高于对照(pYES2转化酵母),结果显示了碱茅的Cu/Zn-SOD基因在酵母表达中,具有提高酵母抗盐碱和耐氧化能力.