Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.     

Quantum dots as a novel immunofluorescent detection system for Cryptosporidium parvum and Giardia lamblia

Semiconductor quantum dot-conjugated antibodies were successfully developed to label Cryptosporidium parvum and Giardia lamblia. This novel fluorescence system exhibited superior photostability, gave 1.5- to 9-fold-higher signal-to-noise ratios than traditional organic dyes in detecting C. parvum, and allowed dual-color detection for C. parvum and G. lamblia.     

Nickel(II) and cobalt(II) complexes of hydroxyl-substituted triazamacrocyclic ligand as potential antitumor agents

The stability constants for the formation of nickel(II) and cobalt(II) complexes of the ligand [1,4,7]triazecan-9-ol (L) were presented. Antitumor activity of two complexes was reported. Nuclei of [NiL]-stimulated BEL-7402 cells clearly exhibited condensation and break down into chromatin clumps typical of apoptosis. Also it exhibited perturbation effects to cell cycle, and optimal induction of apoptosis was found by Flow-Cytometric analysis. But CoL complex did not exhibit introduction effects to BEL-7402 cells apoptosis; and could not perturb cell cycle. NiL and CuL complexes could cleave supercoiled DNA (pBR 322 DNA) to nicked and linear DNA, and DNA of cells treated with NiL or CuL complex was obviously damaged; while CoL complex only could cleave supercoiled DNA (pBR 322 DNA) to nicked DNA, and DNA of cells treated with CoL complex had no significant difference with control.     

Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.     

A probabilistic method to correlate ion pairs with protein thermostability

Recent developments in research on the stability of proteins - specifically, comparisons of the ion pairs of homologous structures - show that ion pairs potentially contribute to the thermostability of proteins. This study proposes a probabilistic Bayesian statistical method to efficiently predict the thermostability of proteins based on the properties of ion pairs. The experimental results suggest that the numbers, types and bond lengths of ion pairs can be used to predict with high accuracy (up to 80%) the thermostability of functionally similar proteins. The predictions have high precision (99%), especially for hyperthermophilic proteins. Results for proteins with differing functions also indicate that the number of ion pairs is related to the thermostability of proteins, and that predictions of thermostability can also be made for proteins with different functions.     

A statistical method for identifying differential gene-gene co-expression patterns

MOTIVATION: To understand cancer etiology, it is important to explore molecular changes in cellular processes from normal state to cancerous state. Because genes interact with each other during cellular processes, carcinogenesis related genes may form differential co-expression patterns with other genes in different cell states. In this study, we develop a statistical method for identifying differential gene-gene co-expression patterns in different cell states. RESULTS: For efficient pattern recognition, we extend the traditional F-statistic and obtain an Expected Conditional F-statistic (ECF-statistic), which incorporates statistical information of location and correlation. We also propose a statistical method for data transformation. Our approach is applied to a microarray gene expression dataset for prostate cancer study. For a gene of interest, our method can select other genes that have differential gene-gene co-expression patterns with this gene in different cell states. The 10 most frequently selected genes, include hepsin, GSTP1 and AMACR, which have recently been proposed to be associated with prostate carcinogenesis. However, genes GSTP1 and AMACR cannot be identified by studying differential gene expression alone. By using tumor suppressor genes TP53, PTEN and RB1, we identify seven genes that also include hepsin, GSTP1 and AMACR. We show that genes associated with cancer may have differential gene-gene expression patterns with many other genes in different cell states. By discovering such patterns, we may be able to identify carcinogenesis related genes.     

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.