Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.     

Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP

The cellular prion protein (PrP^C) consists of a flexible N-terminal tail (FT, aa 23–128) hinged to a membrane-anchored globular domain (GD, aa 129–231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrP_Δ141–225, or “FTgpi”). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

Endothelial cells are activated by angiopoeitin-1 gene transfer and produce coordinated sprouting in vitro and arteriogenesis in vivo

RATIONAL AND OBJECTIVES: Activation of fully differentiated vascular cells using angiogenic genes can lead to phenotypic changes resulting in formation of new blood vessels. We tested whether Ang-1 gene transfer to endothelial cells (EC) activates these cells. METHODS AND RESULTS: EC and SMC were transduced using retroviral or adenoviral vectors to produce Ang-1 or vascular endothelial growth factor (VEGF). EC Tie-2 receptor was phosphorilated by autologous secretion of Ang-1. Transduced EC and SMC sprouting capacity was tested using collagen embedded spheroids assay and capacity to produce arteriogenesis was tested in a hind limb model of ischemia. EC expressing Ang-1 in the presence of SMC expressing VEGF exhibited high levels of sprouting of the two cell types. Flow and numbers of arteries were increased after transduced cells implantation in vivo. CONCLUSIONS: Autologous secretion of Ang-1 by transduced EC resulted in Tie-2 activation and in the presence of SMC expressing VEGF resulted in coordinated sprouting in vitro and increase in flow and number of arteries in vivo.     

中国新疆天山山脉中部微孢衣属的研究

报道了天山山脉中部微孢衣属的3个种类,分别为被膜微孢衣(Acarospora molybdina)、疣微孢衣(Acarospora verruculosa)、戈壁微孢衣(Acarospora gobiensis)。其中被膜微孢衣为中国新记录种。该文对3种微孢衣属的地衣体和子囊盘的形态特征,以及子囊盘、子囊、子囊孢衣和侧丝等解剖特征进行了详细描述,并提供了相关彩色图片。     

新疆艾比湖湿地自然保护区鸟类清单及秋季迁徙数量统计

新疆物种多样性项目组在近10年中多次考察丁艾比湖区(44°50'N,82°50'E,海拔189 m).特别是2007～2009年逐月环湖考察,包括阿拉山口、奎屯河、精河、博尔塔拉河、科克巴斯陶、桑德库木、甘家湖、古尔图等,东西长118 km,南北宽56 km,覆盖6608 km2.采用样线法和点计数法对艾比湖区不同区域鸟的种类和数量进行统计和分析.记录到233种鸟类,是全疆种数的55%,分别隶属于17目53科128属.秋季一次统计到103 875只鸟类.首次发现了卷羽鹈鹕Pelecanus crispus、白头硬尾鸭Oxyura leucocephala、遗鸥Larus relictus和细嘴鸥Larus genei等的聚集地.另有夜鹭、小白额雁、斑背潜鸭、长尾鸭、剑鹆、小滨鹬、细嘴鸥、黄腹鹨等8种为新疆新纪录种.区系以占北种(183种,78.5%)和广布种(49种,21.0%)为主,极少东洋种.艾比湖为中哑鸟类迁徙的重要驿站.     

Ras triggers ataxia-telangiectasia-mutated and Rad-3-related activation and apoptosis through sustained mitogenic signaling

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.     

不同生境胡杨叶片δ~(13)C和δ~(15)N及其对环境因子的响应

以新疆南北疆8个不同生境林龄群体的胡杨叶片为材料,测定幼树和成熟胡杨叶片的天然稳定碳、氮同位素组成值(δ~(13)C、δ~(15)N)以及碳含量、氮含量和比叶面积,分析叶片δ~(13)C、δ~(15)N值与海拔、经纬度、叶片碳氮含量、比叶面积以及水分利用效率之间的相互关系。结果表明:(1)胡杨幼树和成熟林叶片δ~(13)C平均值分别为-27.863‰(-28.776‰～-26.695‰)和-28.230‰(-29.717‰～-26.033‰),不同生境胡杨叶片间的δ~(13)C值具有显著差异(P0.05),并且幼树林叶片δ~(13)C均大于对应成熟林;幼树和成熟林叶片δ~(15)N平均值分别为3.259‰(-1.842‰～9.082‰)和3.651‰(0.798‰～5.779‰)。(2)胡杨幼树和成熟林叶片碳含量平均值分别为46.225‰(44.573‰～49.056‰)和45.720‰(43.226‰～47.349‰),它们叶片氮含量平均值分别为1.708‰(1.327‰～2.116‰)和1.823‰(1.164‰～2.450‰);成熟林叶片碳含量与其δ~(13)C和δ~(15)N值分别呈极显著负相关和极显著正相关关系(P0.01),而其氮含量与δ~(13)C值呈不显著正相关关系(P0.05),与δ~(15)N值呈显著正相关关系。(3)胡杨幼树林叶片的比叶面积平均值(91.565 cm~2/g)小于成熟林叶片(103.141 cm~2/g)。(4)幼树和成熟林胡杨叶片δ~(13)C、δ~(15)N值均与纬度呈极显著正相关关系,幼树林叶片δ~(13)C、δ~(15)N值与海拔也呈极显著正相关关系,幼树林叶片δ~(15)N值与经度也呈显著正相关关系。(5)幼树和成熟胡杨林水分利用率的平均值分别为77.618μmol/mol(68.070～91.069μmol/mol)和72.463μmol/mol(62.809～97.111μmol/mol),不同林龄的胡杨水分利用率均与其叶片δ~(13)C呈极显著正相关关系(P0.001),各生境中于田县(阿日系马扎)的幼树和成熟林叶片具有较高的δ~(13)C值(-26.695‰和-26.033‰)和水分利用效率(91.069和97.111μmol/mol)。     

新疆双峰驼天然单域抗体库的构建及初步鉴定

旨在构建新疆双峰驼天然单域抗体库,及从中快速筛选VHH抗体.采用两种不同的抗原(溶菌酶和cAb-HEWL23)用天然文库进行筛选,成功筛选到了相应的抗体,并融溶菌酶筛选的VHH抗体(A3-1,A4-1和A10-1)进行表达和初步的ELISA检测.结果显示,A3-1和A10-1具有结合溶菌酶的能力.该方法简单方便、省时省力,可以快速从天然单城抗体库中筛选VHH抗体.     

Up-regulation of KISS1 as a novel target of Let-7i in melanoma serves as a potential suppressor of migration and proliferation in vitro

Melanoma is a kind of skin cancer that is begun by the alteration of melanocytes. miRNAs are small non-coding RNA molecules that regulate a variety of biological processes. KISS1, the metastasis-suppressor gene, encodes kisspeptins which inhibits migration and proliferation of cancers. This study was aimed to determine the role of Let-7i and KISS1 in melanoma cell migration and proliferation. At first, the expression of Let-7i and KISS1 was determined in patients with melanoma. In the in vitro part of the study, Let-7i mimics were transfected and the impact of its restoration on target gene expression, proliferation, migration and apoptosis of SK-MEL-3 melanoma cell line was assessed by real-time PCR and Western blotting, MTT assay, wound-healing assay and flow cytometry, respectively. Besides, KISS1 inhibitor siRNA alone and along with Let-7i was transfected to determine their probable correlation. The results revealed that either Let-7i or KISS1 were down-regulated in patients with melanoma. The results obtained from the in vitro part of the study revealed that restoration of Let-7i reduced the expression of metastasis- and proliferation-related target genes. Moreover, it was revealed that up-regulation of Let-7i attenuated migration and proliferation capability of SK-MEL-3 cells. Besides, it was demonstrated that Let-7i restoration induced apoptosis in melanoma cells. More importantly, the KISS1 inhibitor caused a prominent cell migration and proliferation, attenuated by Let-7i re-expression. To sum up, the present study revealed the impressive role of Let-7i restoration along with its correlation with KISS1 on melanoma carcinogenicity which may be applicable in future in vivo studies.