Life history characteristics of the protogynous parrotfish Calotomus japonicus from northwest Kyushu, Japan

In this study, we examined the life history characteristics of the parrotfish Calotomus japonicus, using individuals collected between May 2003 and May 2008 off the Nagasaki Peninsula in northwest Kyushu, Japan. Age determinations were performed using scales. Marginal increment analysis revealed that growth rings were formed annually around July. Growth in both sexes was fitted to the von Bertalanffy growth function (L _∞ = 513, k = 0.28, t ₀ = 0.03, where L _∞ is the theoretical asymptotic total length in mm, k is the growth rate coefficient and t ₀ is the theoretical time at zero length). Observed maximum age for both sexes was 8 years. We also characterized the reproductive biology of this species based on a gonadosomatic index and histological examinations of the gonads. The spawning season extends from July to October, with peak spawning activity occurring during July and August. Fish reach sexual maturity by the second year of life. Females are assumed to be multiple spawners, since we observed specimens with postovulatory follicles in ovaries containing either yolk globule oocytes or migratory nucleus oocytes. All males had secondary testes, which were characterized by the presence of an ovarian lumen structure and sperm sinuses in the gonadal wall. This indicates that all males, irrespective of whether they were initial or terminal phase males, had undergone a sexual transition. Sex change appears to occur during the spawning season, and thereafter sex-changed males are able to fertilize female eggs throughout the remainder of the current spawning season.     

The occurrence of eukaryotic type III glutamine synthetase in the marine diatom Chaetoceros compressum

Glutamine synthetase (GS) has been described as one of the oldest functioning genes and thus a good molecular clock protein. GS is diverged into three distinct forms, type I (GSI), type II (GSII) and type III (GSIII), the last type of which is a member of the most recently discovered family among GSs and thus has been reported from a limited number of prokaryotes. In the present study, we determined the full-length sequence of GSIII from the marine diatom Chaetoceros compressum. The 3′ untranslated region of the diatom GSIII gene was composed of a polyadenylation signal followed by a poly (A)⁺ tail, clearly demonstrating that its mRNA is transcribed from the eukaryotic genome. We also screened available genome databases and identified full-length GSIII sequences from 5 eukaryotic species. These eukaryotic GSIIIs specifically contained regions A–D and a long additional sequence flanking region V toward the C-terminal site, both being specific to GSIII. Phylogenic analysis revealed that eukaryotic GSIIIs are not within a monophyletic relationship with the possible occurrence of lateral gene transfer in GSIII during evolution.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.     

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.     

Retinoic acid activates myogenesis in vivo through Fgf8 signalling

Retinoic acid (RA) has been shown to regulate muscle differentiation in vitro. Here, we have investigated the role of RA signalling during embryonic myogenesis in zebrafish. We have altered RA signalling from gastrulation stages onwards by either inhibiting endogenous RA synthesis using an inhibitor of retinaldehyde dehydrogenases (DEAB) or by addition of exogenous RA. DEAB reduces expression of the myogenic markers myoD and myogenin in somites, whereas RA induces increased expression of these genes and strongly induces premature myoD expression in the presomitic mesoderm (psm). The expression dynamics of myf5 in presomitic and somitic mesoderm suggest that RA promotes muscle differentiation, a role supported by the fact that RA activates expression of fast myosin, while DEAB represses it. We identify Fgf8 as a major relay factor in RA-mediated activation of myogenesis. We show that fgf8 expression in somites and anterior psm is regulated by RA, and find that in the absence of Fgf8 signalling in the acerebellar mutant RA fails to promote myoD expression. We propose that, in the developing embryo, localised synthesis of RA by Raldh2 in the anterior psm and in somites activates fgf8 expression which in turn induces the expression of myogenic genes and fast muscle differentiation.     

Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.     

微生物多糖合成关键基因挖掘的研究进展

随着分子生物学技术的快速发展,功能基因的挖掘在微生物高产多糖合成关键途径研究中变得越来越重要,不断发展的基因挖掘方法和基因组分析工具推进了研究的深入进行.本文主要综述了近年来报道的微生物多糖生物合成途径和多糖合成途径中的关键酶,以及利用多种技术手段和分析软件工具对多糖合成关键基因进行挖掘和验证的相关研究,为微生物多糖合...     

不同种源青檀种子的营养成分及种子活力的差异

以采自安徽（ＡＨ）、山东（ＳＤ）及江苏（ＪＳ）的６个青檀（ＰｔｅｒｏｃｅｌｔｉｓｔａｔａｒｉｎｏｗｉＭａｘｉｍ．）种源种子为材料,测定各种源种子营养成分含量及种子活力。６个种源种子的蛋白质、可溶性糖、淀粉及粗脂肪含量存在较大差异。在种子品质上,６个种源的种子千粒重、浸泡液电导率及简化种子活力指数（ＳＶＩＳ）也存在显著差异。种子中各营养成分含量的高低与ＳＶＩＳ的大小密切相关,复相关系数达０８２２３,以蛋白质含量对ＳＶＩＳ的影响最大,淀粉和粗脂肪含量影响次之,可溶性糖含量影响最小。采用模糊聚类分析法,以种子千粒重、电导率及ＳＶＩＳ为评价指标,可将６个种源的种子品质划分为三大类：第一类包括ＳＤ１、ＡＨ１和ＪＳ１３个种源,种子品质较好;第二类仅为ＡＨ３种源,品质中等;第三类包括ＡＨ２和ＳＤ２２个种源,品质较差。     

A preliminary study of the occurrence of actidione-resistant fungi in sediments of Catalonian river mouths (Spain). I. Keratinolytic fungi and related Onygenales

Sediments from eight river mouths along the Catalonian coast (Spain) were surveyed for keratinolytic fungi and related Onygenales. The actidione plating technique was employed. Of 532 actidione-treated sediment samples, 268 (50.3%) were positive for the fungi. Altogether, 384 fungal strains from 35 species were isolated from the samples. Narasimhella marginospora, Aphanoascus fulvescens, Neoarachnotheca keratinophila with its anamorph Myriodontium keratinophilum, Narasimhella hyalinospora, Beauveria alba, Sporothrix schenckii, Chrysosporium lobatum and Gymnoascus littoralis were the predominant species in sediments. Abundance of N. marginospora was clearly correlated with the degree of water pollution with sewage. This revised version was published online in June 2006 with corrections to the Cover Date.