Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Phylogeographic structure and late Quaternary population history of the Japanese oak Quercus mongolica var. crispula and related species revealed by chloroplast DNA variation

Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnH-psbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (HT) and haplotype diversity within populations (HS) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.     

Degradation of arylarsenic compounds by microorganisms

Microorganisms were not directly accumulated when soil contaminated to about 0.5 mM with diphenylarsinic acid (DPAA) was used as the sole source of carbon. However, using toluene as the carbon source yielded several isolates, which were then used in cultivation with DPAA as the sole source of carbon. By these methods, Kytococcus sedentarius strain NK0508, which can grow in up to 0.038 mM DPAA, was isolated. The toxicity of DPAA retarded the growth of K. sedentarius and the direct accumulation of DPAA-utilizing microorganisms from environmental samples. This strain can utilize about 80% of DPAA and phenylarsonic acid as the sole source of carbon for 3 days. Degradation products of DPAA were determined to be cis, cis, muconate and arsenic acid. When K. sedentarius was cultivated with methylphenylarsinic acid and diphenylmethylarsine, about 90% and 10% degradation of the two compounds, respectively, were observed. Diphenylmethylarsine oxide, possibly synthesized by methylation of DPAA, was detected as one of the transformation products. These results suggest that degradation is initiated by splitting of the phenyl groups from the arylarsenic compounds with subsequent hydroxylation of the phenyl groups and ring opening to yield cis, cis, muconate.     

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.     

Concerted and nonconcerted evolution of the Hsp70 gene superfamily in two sibling species of nematodes

We have identified the Hsp70 gene superfamily of the nematode Caenorhabditis briggsae and investigated the evolution of these genes in comparison with Hsp70 genes from C. elegans, Drosophila, and yeast. The Hsp70 genes are classified into three monophyletic groups according to their subcellular localization, namely, cytoplasm (CYT), endoplasmic reticulum (ER), and mitochondria (MT). The Hsp110 genes can be classified into the polyphyletic CYT group and the monophyletic ER group. The different Hsp70 and Hsp110 groups appeared to evolve following the model of divergent evolution. This model can also explain the evolution of the ER and MT genes. On the other hand, the CYT genes are divided into heat-inducible and constitutively expressed genes. The constitutively expressed genes have evolved more or less following the birth-and-death process, and the rates of gene birth and gene death are different between the two nematode species. By contrast, some heat-inducible genes show an intraspecies phylogenetic clustering. This suggests that they are subject to sequence homogenization resulting from gene conversion-like events. In addition, the heat-inducible genes show high levels of sequence conservation in both intra-species and inter-species comparisons, and in most cases, amino acid sequence similarity is higher than nucleotide sequence similarity. This indicates that purifying selection also plays an important role in maintaining high sequence similarity among paralogous Hsp70 genes. Therefore, we suggest that the CYT heat-inducible genes have been subjected to a combination of purifying selection, birth-and-death process, and gene conversion-like events.     

Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.     

Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.