利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

Effects of Environmental Factors on Byssal Thread Formation in Some Members of the Family Mytilidae from the Sea of Japan

The ability to attach repeatedly to a substrate (glass, boulders, sand) in three common mussel species of the upper sublittoral zone of the Sea of Japan, Grayan's mussel Crenomytilus grayanus, the Korean mussel Mytilus coruscus, and northern horse mussel Modiolus modiolus, was studied under experimental conditions. It was found that during 120 h of the experiment C. grayanus and M. modiolus produced more byssal threads than M. coruscus. A decrease in the water temperature from 20 to 0°C slowed the rate of production of byssal threads down to full passivity in some experimental mollusks. This was more typical of M. coruscus and less typical of C. grayanus. Renewed threads differed in their length, thick, size of the adhesive plate, and strength. M. coruscus formed the shortest, thickest, and strongest threads with rather a large adhesive disk. The observed differences are discussed from the position of morphophysiological adaptations of species for colonization of different natural substrata under contrasting conditions of the upper sublittoral zone.     

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.     

<Emphasis Type="Italic">Medicago truncatula</Emphasis> syntaxin SYP132 defines the symbiosome membrane and infection droplet membrane in root nodules

Symbiotic association of legume plants with rhizobia bacteria culminates in organogenesis of nitrogen-fixing root nodules. In indeterminate nodules, plant cells accommodate rhizobial infection by enclosing each bacterium in a membrane-bound, organelle-like compartment called the symbiosome. Numerous symbiosomes occupy each nodule cell; therefore an enormous amount of membrane material must be delivered to the symbiosome membrane for its development and maintenance. Protein delivery to the symbiosome is thought to rely on the plant secretory system; however, the targeting mechanisms are not well understood. In this study, we report the first in-depth analysis of a syntaxin localized on symbiosome membranes. Syntaxins help define a biochemical identity to each compartment in the plant secretory system and facilitate vesicle docking and fusion. Here, we present biochemical and cytological evidence that the SNARE MtSYP132, a Medicago truncatula homologue of Arabidopsis thaliana Syntaxin of Plants 132, localizes to the symbiosome membrane. Using a specific anti-MtSYP132 peptide antibody, we also show that MtSYP132 localizes to the plasma membrane surrounding infection threads and is most abundant on the infection droplet membrane. These results indicate that MtSYP132 may function in infection thread development or growth and the early stages of symbiosome formation. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Reinforced bioartificial dermis constructed with collagen threads

In this work, a novel type of composite scaffold was designed, which has the suitability of both high biocompatibility and strong mechanical properties, for use in bioartificial dermis applications. The reinforced scaffold consisted of a lyophilized collagen sponge formed around a cross-linked collagen meshwork with an average thread diameter of approximately 55 μm. Fibroblasts were cultured in the reinforced collagen sponge for 7 days, during which time the pores in the sponge became filled with cells that secreted extracellular matrix (ECM) to form a bioartificial dermis. Results of ultimate tensile strength (UTS) measurements and compression tests indicated that the bioartificial dermis formed around the reinforced collagen sponge showed about ten times the strength of the bioartificial dermis formed around a typical collagen sponge (1.5 ± 0.05 vs. 0.15 ± 0.05 and 2.5 ± 0.1 vs. 0.2 ± 0.08 MPa, respectively). As a result, reinforced collagen mesh improved mechanical properties and this technique will be possible to make stronger scaffolds, not only for artificial skin applications but also various artificial tissues, such as synthetic cartilage, bone, and blood vessels.     

PML2‐mediated thread‐like nuclear bodies mark late senescence in Hutchinson–Gilford progeria syndrome

Progerin accumulation disrupts nuclear lamina integrity and causes nuclear structure abnormalities, leading to premature aging, that is, Hutchinson–Gilford progeria syndrome (HGPS). The roles of nuclear subcompartments, such as PML nuclear bodies (PML NBs), in HGPS pathogenesis, are unclear. Here, we show that classical dot‐like PML NBs are reorganized into thread‐like structures in HGPS patient fibroblasts and their presence is associated with late stage of senescence. By co‐immunoprecipitation analysis, we show that farnesylated Progerin interacts with human PML2, which accounts for the formation of thread‐like PML NBs. Specifically, human PML2 but not PML1 overexpression in HGPS cells promotes PML thread development and accelerates senescence. Further immunofluorescence microscopy, immuno‐TRAP, and deep sequencing data suggest that these irregular PML NBs might promote senescence by perturbing NB‐associated DNA repair and gene expression in HGPS cells. These data identify irregular structures of PML NBs in senescent HGPS cells and support that the thread‐like PML NBs might be a novel, morphological, and functional biomarker of late senescence.     

厚壳贻贝Mytilus coruscus足丝盘及足丝的多巴分析

多巴(3,4-1-dihydroxyphenylalanine,DOPA)是贻贝足丝粘附蛋白中的一种特殊的氨基酸,由酪氨酸经羟化后生成,与贻贝足丝粘附蛋白的强粘附性能具有直接联系.目前,已鉴定的多种贻贝足丝蛋白序列中均发现有不同含量的DOPA存在.蛋白中DOPA的定量检测对于了解DOPA在蛋白粘附中的作用以及粘附蛋白的...     

Dynamics of byssal thread production in <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> and <Emphasis Type="Italic">Modiolus modiolus</Emphasis> (Bivalvia) upon reattachment to substrate

It was established that the process of repeated attachment to a substrate in bivalve mollusks Crenomytilus grayanus (Grays mussel) and Modiolus modiolus (the northern horse mussel) involved several successive stages which took in vitro about one month at a water temperature of 19 ± 2°C. Comparison with Grays mussel revealed that the northern horse mussel had a higher rate of byssal thread production and a greater thread number by the end of the complete formation of the byssus complex. The observed differences are explained by the adaptation of mollusks to habitation in different biotopes.Original Russian Text Copyright © 2004 by Biologiya Morya, Selin, Vekhova.