Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.     

CCl4致小鼠肝损伤中几种免疫介质含量变化的研究

本文通过研究CCl4致小鼠肝损伤组织匀浆和血浆一些免疫介质含量的变化以探讨这些免疫介质在CCl4诱发肝损伤过程中作用机制.分别选用30只健康成年小鼠,雌雄各半,随机分成对照组和CCl4负荷组,每组15只.通过腹腔注射CCl4诱发肝损伤后,分别在第2、4、6周检测肝组织匀浆cAMP、cGMP和MDA及血浆IL-2、TNF-α水平的变化.结果显示,在整个实验期内,CCl4组肝组织匀浆cAMP水平均低于或明显低于对照组;cGMP在实验第2周后,高于或显著高于对照组;cAMP/cGMP比值呈现下降趋势,并低于或明显低于对照组;MDA含量明显高于对照组.在整个实验期内,CCl4组血浆IL-2水平下降或显著下降;TNF-α水平则均高于或显著高于对照组.结果提示,CCl4负荷诱发免疫介质cAMP、cGMP、TNF-α和IL-2发生剧烈变化,在介导肝损伤过程中可能起重要作用.     

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots

The fluorescent labeling of biological materials usingsmall-molecule organic dyes is widely employed in bio-logical imaging and clinical diagnosis. Organic fluoro-phores, however, have certain characteristics that limittheir advantages in some applications. These limitationsinclude narrow excitation bands and broad emissionbands with red spectral tails, which make the simultaneousevaluation of several light-emitting probes difficult due tospectral overlap. Also, many organic dyes exhibit highp…     

Structure-function evolution of the Transforming acidic coiled coil genes revealed by analysis of phylogenetically diverse organisms

Background  

Examination of ancient gene families can provide an insight into how the evolution of gene structure can relate to function. Functional homologs of the evolutionarily conserved transforming acidic coiled coil (TACC) gene family are present in organisms from yeast to man. However, correlations between functional interactions and the evolution of these proteins have yet to be determined.     

Two-dimensional mass spectra generated from the analysis of 15N-labeled and unlabeled peptides for efficient protein identification and de novo peptide sequencing

Protein identification has been greatly facilitated by database searches against protein sequences derived from product ion spectra of peptides. This approach is primarily based on the use of fragment ion mass information contained in a MS/MS spectrum. Unambiguous protein identification from a spectrum with low sequence coverage or poor spectral quality can be a major challenge. We present a two-dimensional (2D) mass spectrometric method in which the numbers of nitrogen atoms in the molecular ion and the fragment ions are used to provide additional discriminating power for much improved protein identification and de novo peptide sequencing. The nitrogen number is determined by analyzing the mass difference of corresponding peak pairs in overlaid spectra of (15)N-labeled and unlabeled peptides. These peptides are produced by enzymatic or chemical cleavage of proteins from cells grown in (15)N-enriched and normal media, respectively. It is demonstrated that, using 2D information, i.e., m/z and its associated nitrogen number, this method can, not only confirm protein identification results generated by MS/MS database searching, but also identify peptides that are not possible to identify by database searching alone. Examples are presented of analyzing Escherichia coli K12 extracts that yielded relatively poor MS/MS spectra, presumably from the digests of low abundance proteins, which can still give positive protein identification using this method. Additionally, this 2D MS method can facilitate spectral interpretation for de novo peptide sequencing and identification of posttranslational or other chemical modifications. We envision that this method should be particularly useful for proteome expression profiling of organelles or cells that can be grown in (15)N-enriched media.     

Macrophage-Specific Overexpression of Human Matrix Metalloproteinase-12 in Transgenic Rabbits

Increased matrix metalloproteinase-12 (MMP-12) has been implicated in atherosclerosis and many other inflammatory processes. To define MMP-12 functions in vivo, we generated transgenic rabbits that expressed human (h) MMP-12 gene under the control of a macrophage-specific promoter, the human scavenger receptor promoter. Two transgenic founder rabbits were found to have hMMP-12 transgene integration by Southern blot analysis. hMMP-12 mRNA was expressed in peritoneal and alveolar macrophages, and in tissues enriched in macrophages in transgenic rabbits. High levels of hMMP-12 protein were detected in the conditioned media of cultured peritoneal and alveolar macrophages from transgenic rabbits. Zymography showed that hMMP-12 secreted from macrophages possessed enzymatic activity toward β-casein. To evaluate the expression of hMMP-12 in inflammatory sites, we used carrageenan-induced granulomas as an in vivo model for tissue macrophages and foam cells. Granuloma size in transgenic rabbits was significantly increased compared to that in control rabbits, and histological examination revealed that granulomas of transgenic rabbits were enriched in macrophages associated with increased hMMP-12 expression. We believe that this transgenic rabbit model with increased expression of hMMP-12 may become a useful model for further mechanistic studies of MMP-12 in inflammatory diseases and cancer invasion; it is also an ideal model for testing the in vivo action of MMP-12 inhibitors.     

水葫芦苗(Halerpestes cymbalaris)的生长特征研究

以调查统计的方法在中国科学院海北定位站研究了高寒湿地植物水葫芦苗无性系的生长特征、形态特征以及能量分配规律。结果表明：匍匐茎只有1条的水葫芦苗最多,占35．29％,匍匐茎有4条的水葫芦苗只占8．82％。同一水葫芦苗无性系中,随着匍匐茎数目的增多,分株数、间隔子数、茎总长和匍匐茎比节问重变小。分株一般在第一级最高,末级较低;第1条匍匐茎的间隔子较长。随水葫芦苗匍匐茎数目的增多,用于无性繁殖的分株干重比例逐渐增加,用于有性繁殖的花的干重比例下降。水葫芦苗无性系这种生长特征、形态特征以及能量分配规律是其生物—生态学特性和所处高寒湿地生境共同决定的。