斑蝥素对粘虫几种代谢酶及多酚氧化酶的影响

为进一步探讨斑蝥素的杀虫作用机理, 本研究采用叶碟饲喂法处理粘虫Mythimna separata（Walker）5龄幼虫, 测定了饲喂处理后6, 12, 24, 36和48 h试虫体内羧酸酯酶(CarE)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(ALP)、谷胱甘肽 S-转移酶(GST)和细胞色素P450 Ο-脱甲基酶及多酚氧化酶(PPO)的活性。结果表明: 斑蝥素可显著激活羧酸酯酶, 处理后48 h酶比活力最大, 为同期对照的1.68倍; 酸性磷酸酯酶在处理后6和12 h活性变化不明显, 处理24 h后逐渐被激活, 且与对照差异显著(P＜0.05); 明显抑制碱性磷酸酯酶, 且随着处理时间的延长, 抑制作用增强; 对Ο-脱甲基酶表现为先抑制后激活的趋势; 谷胱甘肽S-转移酶表现出先激活后抑制的影响; 离体活体条件下均可显著抑制PPO活性。可见, 斑蝥素可明显影响昆虫的代谢酶系, 且其对碱性磷酸酶和多酚氧化酶的抑制作用可能与其毒杀作用有关。     

Nutritional composition and digestibility by ocelots (Leopardus pardalis) of whole animals and a commercial diet

Felids are adapted to eat whole prey, but in North American zoos are usually fed processed diets based on muscle meat. We analyzed proximate nutrient composition and digestibility by ocelots of a commercial processed diet and whole animals of five species. The processed diet did not differ significantly from the whole animals in proximate composition, although it was at one end of the range of results for all nutrients. Domestic chicks were significantly lower than all other dietary items tested in digestibility of energy and fat, and lower than rabbits and quail in digestibility of dry matter. There were no other significant differences. These results suggest that the commercial diet tested provides an appropriate nutritional environment for ocelots with respect to proximate constituents. Studies of vitamin and mineral composition and digestibility and comparisons to wild prey species should be conducted to permit a full evaluation. Zoo Biol 29:753–759, 2010. © 2009 Wiley‐Liss, Inc.     

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.     

The crystal structure of the Y140F mutant of ADP-l-glycero-d-manno-heptose 6-epimerase bound to ADP-��-d-mannose suggests a one base mechanism

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L ‐glycero‐D ‐manno‐heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6″ position converting ADP‐D ‐glycero‐D ‐manno‐heptose into ADP‐L ‐glycero‐D ‐manno‐heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP‐L ‐glycero‐D ‐manno‐heptose 6‐epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP‐β‐D ‐mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.     

利用黑曲霉固态发酵啤酒糟生产饲料复合酶的研究

以啤酒糟为主要基质,利用黑曲霉固态发酵生产酸性蛋白酶、木聚糖酶和纤维素酶等多种饲料复合酶,研究了黑曲霉固态发酵培养基组成对复合酶酶活的影响,确定最优培养基配方为:啤酒糟75%,麸皮25%,硫酸铵1%,KH_2PO_4 0.2%,MnSO_4 0.1%、ZnSO_4 0.2%,料水比1:2。在适宜的发酵条件下,经30℃发酵5 d,烘干后得到的复合酶制剂中,具有多种酶活性(以干基计)。其中酸性蛋白酶活力3 800 U/g,木聚糖酶活力12 00 U/g和纤维素酶活力18 U/g。     

Neutrophil Gelatinase-Associated Lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase <Subscript>1, 2</Subscript>

Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase _{(1, 2)} and superoxide dismutase _{(1, 2)} which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD_{1, 2} were compared with appropriate controls by RT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD_{1, 2} enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD₁ and SOD₂ were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD₁ and SOD₂ and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1and SOD_{1, 2}.     

芳香聚酮早期后修饰环化酶的结构分类和功能分析

聚酮是一类结构和生物活性多样的天然产物,根据结构特点可以分为芳香聚酮和复合聚酮两大类。芳香聚酮环化酶是芳香聚酮生物合成过程中一种非常重要的早期后修饰酶,是决定芳香聚酮骨架结构的主要影响因素。根据序列和结构的相似性,芳香聚酮环化酶可以分为不同的种类。本文主要对其中3类芳香聚酮环化酶结构和功能进行了简要总结,从晶体结构、催化反应和催化机制等方面对它们进行了分类描述和功能分析,并结合自己实验室工作介绍了杰多霉素B环化酶催化机制的研究方法。     

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.