甜菜坏死黄脉病毒的分离提纯及其生物化学特性的研究

本研究工作中,建立了一个有效的甜菜坏死黄脉病毒的分离提纯程序,解决了该病毒粒体易于聚集难以提纯的问题,其操作要点是,(1)通过Sepharose 2B柱层析代替超离心,有效地除去一些小分子量核酸杂质;(2)经PEG再次沉淀浓缩后,调整pH至酸牲(pH3.0),使病毒充分悬浮以减少凝聚;(3)在病毒等电点(pH4.8～4.9)条件下,进一步沉淀以纯化病毒。根据病毒提取物的OD260/OD280比值,算出核酸含量约4.5％。核酸电泳出现4条带,分子量分别为:2.25×10~(?),1.8×10~(?),1.05×10~(?),0.75×10~(?)道尔顿。病毒提取物经超速离心出现4个界面,沉淀系数分别为,200.8S,165S,125.8S,100S。说明甜菜坏死黄脉病毒可能是4组分病毒粒体。病毒粒体含一蛋白亚基,分子量约为2.05±0.05×10~4道尔顿,由16种共199个氨基酸组成。     

Sustained expression of MCP-1 induced low wall shear stress loading in conjunction with turbulent flow on endothelial cells of intracranial aneurysm

Shear stress was reported to regulate the expression of AC007362, but its underlying mechanisms remain to be explored. In this study, to isolate endothelial cells of blood vessels, unruptured and ruptured intracranial aneurysm (IA) tissues were collected from IA patients. Subsequently, quantitative real-time PCR (qRT-PCR), Western blot and luciferase assay were performed to investigate the relationships between AC007362, miRNAs-493 and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Reduced representation bisulphite sequencing (RRBS) was performed to assess the level of DNA methylation in AC007362 promoter. Accordingly, AC007362 and MCP-1 were significantly up-regulated while miR-493 was significantly down-regulated in HUVECs exposed to shear stress. AC007362 could suppress the miR-493 expression and elevate the MCP-1 expression, and miR-493 was shown to respectively target AC007362 and MCP-1. Moreover, shear stress in HUVECs led to the down-regulated DNA methyltransferase 1 (DNMT1), as well as the decreased DNA methylation level of AC007362 promoter. Similar results were also observed in ruptured IA tissues when compared with unruptured IA tissues. In conclusion, this study presented a deep insight into the operation of the regulatory network of AC007362, miR-493 and MCP-1 upon shear stress. Under shear stress, the expression of AC007362 was enhanced by the inhibited promoter DNA methylation, while the expression of MCP-1 was enhanced by sponging the expression of miR-493.     

Activation of PPAR-β/δ Attenuates Brain Injury by Suppressing Inflammation and Apoptosis in a Collagenase-Induced Intracerebral Hemorrhage Mouse Model

Neurochemical Research - Brain injury has been proposed as the major cause of the poor outcomes associated with intracerebral hemorrhage (ICH). Emerging evidence indicates that the nuclear...     

Molecular evolution and functional divergence of the Ca(2+) sensor protein in store-operated Ca(2+) entry: stromal interaction molecule

Receptor-mediated Ca(2+) signaling in many non-excitable cells initially induces Ca(2+) release from intracellular Ca(2+) stores, followed by Ca(2+) influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs) function as the Ca(2+) sensor to detect changes of Ca(2+) content in the intracellular Ca(2+) stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+) binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.     

垦殖对新疆绿洲农田土壤有机碳组分及团聚体稳定性的影响

土壤有机碳是土壤质量变化的重要指标,土壤活性有机碳组分在土壤质量变化方面发挥重要作用。采用有机碳分组技术,研究了干旱荒漠区自然土壤开垦对绿洲农田土壤有机碳活性组分及团聚体稳定性的影响。结果表明:低有机碳含量的自然土壤垦殖后,有利于干旱荒漠区绿洲棉田土壤有机碳的积累,且垦殖(0-5a)增加显著,年均增加在0.65gkg-1以上,上升幅度为76%-286%,5a后维持在相对平衡的水平;土壤活性有机碳、轻组有机碳在垦殖0-5a显著增加,平均增加72%和99%,5a后下降;颗粒有机碳则表现出垦殖0-10a明显增加,增加在275%以上,10a后下降;土壤水稳性团聚体含量随垦殖年限的延长显著增加,0-20a内较自然土壤提高了75%。垦殖可能是干旱区绿洲农田潜在碳汇的重要影响因素;但随垦殖年限延长,土壤有机碳活性组分下降,土壤质量又存在一定的退化风险。     

Killing effect of interleukin-13 receptor alpha 2 (IL-13Rα2) sensitized DC-CTL cells on human glioblastoma U251 cells

Recent studies show that IL-13Rα2, a brain tumor-associated antigen for IL-13, may play a role in immunotherapy for glioblastoma. Thus, we stimulated the lymphocyte by monocyte-derived dendritic cells (DCs). The DCs were pulsed with IL-13Rα2 in vitro and then co-cultured with lymphocytes. After inducing cytotoxic T cells (CTLs) and co-culturing with U₂₅₁ cells for 24 h in 96 wells, Cell Count Kit-8 (CCK-8) was added to every well equally. The optical density (OD) value was detected and recorded after 2 h. The DCs efficiently presented the antigen to the CTLs, resulting in CTLs activation and proliferation. The induced CTLs showed specific cytotoxic against U₂₅₁ cells (P < 0.01). The results demonstrated that IL-13Rα2 induced CTLs could kill glioma U₂₅₁ in vitro, which suggests that IL-13 Rα2 might have such an impact in vivo and thus recombinant IL-13Ra2 protein might be used as an anti-tumor vaccine, providing a promising new strategy for the treatment of brain malignant gliomas.     

Influence of substituents on the structures of hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates

By changing the substituents on 1,2,4-triazole ring, six novel organic-inorganic hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates, [{Cu₄(L)_x}Mo₈O₂₆] (L = 3,5-diamino-1,2,4-triazole (datrz) and x = 4 for 1; L = 3-amino-1,2,4-triazole (3atrz) and x = 4 for 2; L = 3,5-dimethyl-1,2,4-triazole (dmtrz) and x = 4 for 3; L = 3,5-dimethyl-4-amino-1,2,4-triazole (dmatrz) and x = 6 for 4; L = 3,5-diethyl-4-amino-1,2,4-triazole (deatrz) and x = 4 for 5; L = 3,5-di(n-propyl)-4-amino-1,2,4-triazole (dpatrz) and x = 3 for 6), were obtained. The tetranuclear Cu(I) cluster in compound 1 acts as charge-compensating unit, which is the first polynuclear metal 1,2,4-triazole structure only with N1, N2 bridging mode. Compounds 2, 4, 5 and 6 are of polymeric 1D chains and 3 is of a 2D layer structure. In 2, three distinct Cu(I)-coordination geometries, distorted tetrahedral, T-shaped and V-shaped linear Cu(I), are observed in the same structure. The first extended hybrid structure constructed by δ-octamolybdates is founded in 4. A novel [Mo₈O₂₆]⁴⁻ anion is found in 5, which contains only three crystallographically independent Mo atoms. In compounds 5 and 6, terminal oxo groups of octamolybdate cluster act as μ₃-oxo bridges to link the copper(I) coordination complexes; such an unusual linking manner is unique in the coordination chemistry of octamolybdates with transition metal fragments. The influences of substituent on the structures of the tetranuclear units are also discussed in details.