Single-step Purification by Lectin Affinity and Deglycosylation Analysis of Recombinant Human and Porcine Deoxyribonucleases I Expressed in COS-7 Cells

Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.     

Mutational analysis of a catalytically important loop containing active site and substrate-binding site in Escherichia coli phytase AppA

A phytase from Escherichia coli, AppA, has been the target of protein engineering to reduce the amount of undigested phosphates from livestock manure by making phosphorous from phytic acid available as a nutrient. To understand the contribution of each amino acid in the active site loop to the AppA activity, alanine and glycine scanning mutagenesis was undertaken. The results of phytase activity assay demonstrated loss of activity by mutations at charged residues within the conserved motif, supporting their importance in catalytic activity. In contrast, both conserved, non-polar residues and non-conserved residues tended to be tolerant to Ala and/or Gly mutations. Correlation analyses of chemical/structural characteristics of each mutation site against mutant activity revealed that the loop residues located closer to the substrate have greater contribution to the activity of AppA. These results may be useful in efficiently engineering AppA to improve its catalytic activity.

Abbreviations: AppA: pH 2.5 acid phosphatase; CSU: contacts of structural units; HAPs: histidine acid phosphatases; SASA: solvent accessible surface area; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSM: site-saturation mutagenesis; WT: wild type     

The method of mouse embryoid body establishment affects structure and developmental gene expression

To investigate formation of the three primary germ layers in mouse embryoid bodies (EBs), we observed changes in structure and gene expression over a 7-day culture period. We compared these changes using two methods for EB formation: hanging drop (HD) and static suspension culture (SSC). Light microscopy showed that a stratified columnar epithelial layer developed on the surface of EBs formed using the HD method. From Day 3 in culture, ultrastructural changes occurred in the aligned cellular membranes. Condensation of actin filaments was followed by formation of complicated adherent junctions and dilatation of intercellular canaliculi containing well-developed microvilli. These changes were more marked in EBs formed by the HD method than the SSC method. On Day 5 of culture, Brachyury gene expression, a marker for mesoderm formation, was detected only with the HD method. Nestin, an ectoderm marker, and Foxa2, an endoderm marker, were expressed with both methods. These results suggest that in EBs formed with the HD method, actin formation and Brachyury gene expression mark the transition from two to three primary germ layers. Additionally, the HD method promotes more rapid and complete development of mouse EBs than does the SSC method. While the SSC method is simple and easy to use, it needs improvement to form more complete EBs.     

Enhancement of phagocytic activity of macrophage-like cells by pyrogallol-type green tea polyphenols through caspase signaling pathways

We investigated the phagocytosis-enhancing activity of green tea polyphenols, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) catechin (+C) and strictinin, using VD3-differentiated HL60 cells. EGCG, EGC, ECG and strictinin, but not EC and +C, increased the phagocytic activity of macrophage-like cells, and a caspase inhibitor significantly inhibited phagocytic activities. These results suggest that the pyrogallol-type structure in green tea polyphenols may be important for enhancement of the phagocytic activity through caspase signaling pathways.     

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to ¹H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.     

10-Year risk of colorectal cancer: Development and validation of a prediction model in middle-aged Japanese men

Background: To estimate an individual's probability of developing colorectal cancer (CRC) may aid health professionals and individuals in improving lifestyle behaviors or deciding the screening regimens. As fewer studies on cancer risk prediction were seen so far, we initially developed an assessment tool with synthesizing key information from a variety of CRC risk factors through a large population-based cohort study. Method: The prediction model was derived from 28,115 men in the Japan Public Health Center-based (JPHC) Prospective Study Cohort II (follow-up: 1993–2005), with risk factors selected by Cox proportion hazard regression. 18,256 men in the JPHC Study Cohort I (follow-up: 1995–2005) were used to evaluate the model's performance. Results: 543 and 398 CRCs were diagnosed during the follow-up period in Cohorts II and I, respectively. The prediction model, including age, BMI, alcohol consumption, smoking status, and the daily physical activity level, showed modest discrimination ability for CRC (C = 0.70; 95% confidential interval, 0.68–0.72) in Cohort II and well calibrated in Cohort I (Hosmer–Lemeshow χ² = 14.2, P = 0.08). Conclusion: The 10-year CRC risk prediction model may be used to estimate CRC risk in Japanese men. It may also play a role in the promotion of CRC prevention strategies.     

Molecular characterization of imipenem-resistant Pseudomonas aeruginosa in Hiroshima, Japan

Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.     

In vitro establishment of tumorigenic human B-lymphoblastoid cell lines transformed by Epstein-Barr virus

We studied tumorigenic and phenotypic characteristics of pre- and postimmortal human B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV): preimmortal LCLs showed low telomerase activity and a normal diploid karyotype while postimmortal LCLs showed much higher telomerase activity and maintained a clonal aneuploidic state. Among five postimmortal LCLs tested, LCLs N0005 and N6803 formed colonies in agar medium and showed marked aneuploidy, and N6803 was transplantable into nude mice indicating that it had a complete malignant phenotype, but all preimmortal LCLs and the remaining three postimmortal LCLs lacked these characteristics. The products of tumor suppresser genes, p16(INK4A) and pRb, were downregulated in these two LCLs, and the p53 gene was mutated in N0005 LCL. We believe these results showed for the first time that some postimmortal EBV-transformed LCLs can become tumorigenic, contrary to previous reports, and that these LCLs provide an in vitro model of tumorigenesis induced by EBV.     

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.