Allele-specific targeting of hsa-miR-657 to human IGF2R creates a potential mechanism underlying the association of ACAA-insertion/deletion polymorphism with type 2 diabetes

The biological mechanism of a recent discovered association of type 2 diabetes with the ACAA-insertion/deletion polymorphism at the 3′UTR of the IGF2R gene has remained unclear. A very recently emerging novel polymorphic control layer by microRNAs (miRNAs) makes it possible to elucidate this issue. In this study, a prediction by web tools MicroInspector and miRanda demonstrated that DNA sequence polymorphism (DSPs) ACAA-insertion/deletion in IGF2R 3′UTR is located within the hsa-miR-657 and hsa-miR-453 binding sites. And luciferase reporter assay revealed that hsa-miR-657 acts directly at the 3′UTR of the IGF2R. Furthermore, ACAA-deletion exerted a further repression compared with ACAA-insertion, indicating that hsa-miR-657 regulates IGF2R gene expression in a polymorphic control manner. Importantly, we also demonstrated that hsa-miR-657 can translationally regulate the IGF2R expression levels in Hep G2 cells. Thus, our findings testify the possibility that the ACAA-insertion/deletion polymorphism may result in the change of IGF2R expression levels at least in part by hsa-miR-657-mediated regulation, contributing to the elucidation for the pathogenesis of type 2 diabetes and raise the possibility that miRNAs or in combination with functional DNA sequence polymorphism may be valuable in the treatment of human type 2 diabetes.     

载脂蛋白E在神经系统中的作用

载脂蛋白Ｅ（ａｐｏＥ）在中枢神经系统（ＣＮＳ）生长发育,成熟衰老和损伤修复过程中发挥重要作用。其分子机制是：（１）稳定神经细胞骨架系统;（２）通过ａｐｏＥ受体途径调节神经细胞中胆固醇脂的运输和突触末梢的再生;（３）调控神经元之间及神经细胞与介质之间的相互作用;（４）调节神经细胞的Ｃａ＾２＋离子的平衡。     

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.     

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.     

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.     

瘤胃降解粗纤维产甲烷的厌氧真菌与甲烷菌共培养物的分离鉴定

【目的】本试验从瘤胃中分离鉴定降解粗纤维产甲烷的厌氧真菌与甲烷菌共培养物,为深入探究甲烷菌对厌氧真菌代谢途径的影响及相关调节机制奠定基础。【方法】利用厌氧滚管技术从荷斯坦奶牛瘤胃内容物中分离厌氧真菌与甲烷菌共培养物,通过形态学观察和DAPI染色以及甲烷菌16S rRNA基因序列分析方法分别对厌氧真菌及甲烷菌进行鉴定。【结果】从荷斯坦奶牛瘤胃中共分离到28株厌氧真菌与甲烷菌共培养物。共培养物中的厌氧真菌均为单中心菌株,分别属于Piromyces,Neocallimastix和Caeomyces属,所占百分比为53.57%,42.86%及3.57%。甲烷菌16S rRNA基因序列分析结果表明,共培养物中的甲烷菌均为甲烷短杆菌。本研究共获得四种不同的厌氧真菌与甲烷菌组合,分别为Piromyces/类Methanobrevibacter olleyae菌株,Neocallimastix/类Methanobrevibacter olleyae菌株,Neocallimastix/类Methanobrevibacter thaueri菌株及Caecomyces/类Methanobrevibacter olleyae菌株,分别占总数的53.57%,39.29%,3.57%及3.57%。【结论】分离得到的28株厌氧真菌和甲烷菌共培养物中,占优势的为具有丰富丝状假根的厌氧真菌Piromyces和Neocallimastix以及类Methanobrevibacter olleyae属的甲烷短杆菌。本研究为进一步研究瘤胃内厌氧真菌与甲烷菌相互代谢关系奠定基础。     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Binding and Uptake of RGD-Containing Ligands to Cellular α v β 3 Integrins

The cyclic peptide, cRGDf[N(me)]V, binds to the α _v β ₃ integrin and can disrupt binding of the integrin to its natural ligands in the extracellular matrix. In this work, the ability of a water-soluble, fluorescently labeled variant of the RGD-containing peptide (cRGDfK-488) to bind to integrins on human umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis was characterized. This information was compared to the binding and uptake properties of an α _v β ₃ integrin-specific monoclonal antibody, LM609X. The specificity of the RGD-containing peptide is assessed by comparison with control peptide that does not bind to the α _v β ₃ integrin, cRADfK-488. Using a high purity construct, it is shown that the RGD ligand exhibits dissociation constants in the micromolar range whereas LM609X exhibits dissociation constants in the nanomolar range. However, the RGD ligand showed greater uptake following incubation at temperatures which permit endocytosis. A 7.4-fold increase in uptake of the RGD peptide was observed following a 1 h incubation with HUVEC at 37°C (an endocytosis permissive temperature), as compared to that at 4°C (an endocytosis prohibitive temperature). In contrast, only a 1.9-fold increase in cell-associated fluorescence was observed for similar incubations with LM609X. Results from fluorescence microscopy supports the notion that the RGD peptide is rapidly endocytosed at 37°C as compared to LM609X. These results are discussed with regard to previous work indicating that RGD ligands enter cells by integrin-independent pathways. These studies provide well-controlled measures of how RGD ligands stimulate endocytosis. This may be of considerable interest for intracellular delivery of ligand-associated drugs in anti-angiogenic applications.