Isolation of a bacterium that causes anaaki disease of the red algae Porphyra yezoensis

Anaaki disease causes severe damage to the red algae Porphyra yezoensis from which the Japanese traditional food 'nori'is produced. The causative agent of anaaki disease was isolated by several repeats of single-colony isolation and infection experiments, and was identified as Flavobacterium sp. LAD-1. The bacterium showed hydrolytic activity toward porphyran but not toward other polysaccharides composing the thallus of Porphyra , such as β-1,3-xylan or β-1,4-mannan. The bacterium also showed β-D-galactosidase activity.     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line,Pv11

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5–34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

     

Induction of cell cycle progression by adenovirus E1A gene 13S- and 12S-mRNA products in quiescent rat cells.

Rat 3Y1 cell lines that express either adenovirus type 12 E1A 13S mRNA or 12S mRNA in response to dexamethasone treatment were established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. These cell lines were growth arrested, and the induction of cell cycle progression was analyzed by flow cytometry after switch on of the cDNA by the addition of dexamethasone. The results indicate that the 13S- or 12S-mRNA product alone has the ability to cause progression of the cell cycle at a similar rate. The simultaneous addition of epidermal growth factor accelerated the rate of cell cycle progression in the transition from the G0/G1 phase to the S phase.     

Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.     

High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh

In Bangladesh, despite the official introduction of artemisinin combination therapy in 2004, chloroquine+sulfadoxine/pyrimethamine has been used for the treatment of uncomplicated malaria. To assess the distribution of pfcrt, pfmdr1, dhfr, and dhps genotypes in Plasmodium falciparum, we conducted hospital- and community-based surveys in Bandarban, Bangladesh (near the border with Myanmar) in 2007 and 2008. Using nested PCR followed by digestion, 139 P. falciparum isolates were genotyped. We found fixation of a mutation at position 76 in pfcrt and low prevalence of a mutation at position 86 in pfmdr1. In dhfr, the highest pyrimethamine resistant genotype quadruple mutant was found in 19% of isolates, which is significantly higher prevalence than reported in a previous study in Khagrachari (1%) in 2002. Microsatellite haplotypes flanking dhfr of the quadruple mutants in Bangladesh were identical or very similar to those found in Thailand and Cambodia, indicating a common origin for the mutant in these countries. These observations suggest that the higher prevalence of the dhfr quadruple mutant in Bandarban is because of parasite migration from Myanmar. However, continuous use of sulfadoxine/pyrimethamine would have also played a role through selection for the dhfr quadruple mutant. These results indicate an urgent need to collect molecular epidemiological information regarding dhfr and dhps genes, and a review of current sulfadoxine/pyrimethamine usage with the aim of avoiding the widespread distribution of high levels of resistant parasites in Bangladesh.     

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.     

Antigen receptor editing in anti-DNA transitional B cells deficient for surface IgM

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM(-/low)). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM(-/low) anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vlambdax) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgM(low) transitional splenic B cells of wild-type mice.     

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid in vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.     

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.