链霉菌降解角蛋白的生化机制研究

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0．01mol／L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同作用下逐步水解成多肽、寡肽和游离氨基酸,使角蛋白彻底降解。在角蛋白降解过程中,角蛋白中的硫也随之转化成巯基化合物,H2S和硫酸盐3种含硫化合物存在于降解产物中。     

Recent progress in research on insect histone deacetylases (HDACs)

组蛋白乙酰化修饰是一种重要的蛋白质翻译后修饰方式,由组蛋白乙酰基转移酶HATs和组蛋白去乙酰化酶HDACs共同调节.昆虫HDACs蛋白家族根据其同源性和结构的不同共分为4类,各昆虫物种之间既具有较高的保守同源性,同时也表现出一定的物种特异性.HDACs主要参与昆虫的胚胎发育、体节形成、寿命和神经行为等方面的调节.本文从HDACs蛋白的种类、系统发育、生理功能等方面展开,介绍了近年来国内外昆虫HDACs领域的最新研究进展,以期对研究昆虫表型可塑性调节机制以及探索新的害虫防治方法提供借鉴.     

思茅松毛虫四龄幼虫肠道好氧细菌的ARDRA分析及鉴定

从思茅松毛虫Dendrolimu kikuchii Matsumura4龄幼虫肠道环境中分离、纯化、培养,获得11株好氧细菌菌株。以细菌基因组DNA为模板,用细菌通用引物(27f、1492r)对模板进行PCR扩增,并用4种限制性内切酶HaeⅢ、HindⅢ、HinfⅠ、TaqⅠ对PCR产物进行ARDRA(amplified rDNA restriction analysis)多态性分析,在84%的相似水平上可分成6大类群,多样性丰富。对6大类群的代表菌株进行16SrDNA序列测定,分析表明:分离到的11株好氧细菌中4N05、4N08和4N11归属克雷伯氏杆菌属(Klebsiellasp.),4N02归属Lysinibacillus属;4N03和4N09确定到种,分别是γ-变形杆菌(Gamma proteobacterium)和枯草芽孢杆菌(Bacillus subtilis strain);4N04、4N06、4N07和4N01、4N10也可以确定到种,其中前3株是短短芽孢杆菌(Brevibacillus brevisstrain),后2株是沼泽短芽孢杆菌(Brevibacillus limnophilusstrain),并且这5株菌都归属短芽孢杆菌属(Brevibacillus sp.)。通过研究,既可以为松毛虫的生物防治提供微生态理论依据,又丰富了微生物资源库。     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells

G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.     

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.     

Complete Genome Sequence of Staphylococcus aureus Bacteriophage GH15

GH15 is a polyvalent phage that shows activity against a wide range of Staphylococcus aureus strains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.     

Single-Stranded siRNAs Activate RNAi in Animals

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in?vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.