"Threading" of beta-sheet peptides via radical polymerization

A nonamer peptide containing a diene group in the center of the sequence was synthesized. When the peptide forms an antiparallel beta-sheet, the diene groups align ca. 5 A apart on the beta-sheet. The diene groups successfully photopolymerized without distorting the beta-sheet structure. The obtained beta-sheet showed high stability against acid denaturation and addition of 1,1,1,3,3,3-hexafluoroisopropanol.     

Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI

Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in Tm and 12.5 kJ/mol in DeltaG(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased DeltaCp value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Diverse DNA modification in marine prokaryotic and viral communities

DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent ‘metaepigenomic’ analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.     

Intracellular signaling of gp34, the OX40 ligand: induction of c-jun and c-fos mRNA expression through gp34 upon binding of its receptor, OX40.

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.     

An external ion microbeam for studies of biological samples

A new external ion-beam system was developed and combined with a light ion-microbeam system in JAERI Takasaki. The system is designed for micro-particle-induced X-ray emission analysis of biological samples in air environment with 1 Μm spatial resolution. One of the most serious problems in keeping such a high spatial resolution is multiple scattering in a beam exit window. Thin Kapton film (7.5 Μm thick) was adopted as the exit window as well as a sample backing foil to minimize the distance between the film and samples. The lifetime of the foil under ion irradiation and spatial resolution of the external microbeam were investigated. The results shows that the film can endure sufficient long-time irradiation to take elemental maps and the resolution can be kept nearly 1 Μm.     

Chromosomal assignment of the chicken NP220 gene encoding a large DNA-binding nuclear protein and its targeted disruption in chicken DT40 cells

NP220s form a family of DNA-binding nuclear proteinsoriginally found in human cell lines. Four isoformsof NP220 are produced in humans and mice probably byalternative splicing of two sequence units. Theyhave RNA-recognition and arginine/serine rich motivescommonly found in many nuclear proteins important forpre-mRNA processing. In order to analyze the functionof NP220s, its gene was disrupted in chicken cell lineDT40. For this, gemomic DNA clone of chicken NP220was isolated and the gene was localized tochromosome 4q2.7–q2.8. Chicken NP220 conserves MH1and zinc finger-like motives found in human and mouseNP220s. Despite its expression as a mRNA of 6 kb inwild type DT40 cells, disruption of the NP220gene did not impair the growth of DT40 cells.     

Bioremediation technologies for treatment of PAH-contaminated soil and strategies to enhance process efficiency

The complex and diverse structural configurations of polycyclic aromatic hydrocarbons (PAHs), combined with their low bioavailability, hydrophobic nature, strong sorption phenomena, and high persistence in soil makes the design of effective bioremediation methodologies a challenge. The multi-phasic nature of the bioremediation process, restricted mass transfer and non-availability of degrading soil microflora further compound the problem. In this direction, this communication presents a focused review of bioremediation technologies used recently for the treatment of PAH-contaminated soils. The specific roles of important factors affecting bioremediation process efficiency are discussed. Finally some of the recently used strategies to enhance bioremediation process efficiency, including bioaugmentation, biostimulation, rhizoremediation, the use of chemotaxins, the biomimetic catalytic system approach, and integrated techniques, are reviewed.     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.