Phylogeography of Blacktip Grouper,Epinephelus fasciatus (Perciformes: Serranidae), and influence of the Kuroshio Current on cryptic lineages and genetic population structure

To investigate the influence of the Kuroshio Current on the high diversity of marine fishes in Japanese waters, the intraspecific phylogeographic structure of Blacktip Grouper, Epinephelus fasciatus, was determined. The genetic analysis of E. fasciatus indicated three intraspecific mtDNA lineages representing different evolutionary histories: the first lineage differentiated in Japanese waters during a long period of fluctuations of the ancient Kuroshio Current, the second lineage, widely distributed in the tropical western Pacific, was transported to Japanese waters by the Kuroshio Current and the third lineage was distributed primarily around the Ogasawara (Bonin) Islands. Present-day sympatric distributions of the three lineages, characterized by different ratios of such individuals at each geographic site, indicated a complex genetic pattern that was classified into three demographic groups, the dispersal and gene flows of which were strongly influenced by the Kuroshio Current and factors such as countercurrent and island arc. Genetic breaks in E. fasciatus populations were congruent with other fish faunal boundaries in Japanese waters.     

Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.     

L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

Stable generation of serum‐ and feeder‐free embryonic stem cell‐derived mice with full germline‐competency by using a GSK3 specific inhibitor

C57BL/6 (B6)‐derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well‐defined genetic background because of poor developmental potential. We newly established serum‐ and feeder‐free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6‐bromoindirubin‐3′‐oxime (BIO), a glycogen synthase kinase‐3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight‐cell‐stage diploid embryo, we stably generated germline‐competent ES‐derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum‐ and feeder‐free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background. genesis 47:414–422, 2009. © 2009 Wiley‐Liss, Inc.     

Parvulin (Par14), a Peptidyl-Prolyl cis-trans Isomerase, Is a Novel rRNA Processing Factor That Evolved in the Metazoan Lineage

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.Peptidyl-prolyl cis-trans isomerases (PPIases)¹ catalyze the rotation about the peptide bond on the amino-terminal side of proline, a step that can be rate-limiting for the folding of newly synthesized proteins (1). PPIases also have the ability to bind many proteins, thereby acting as chaperones; thus, they are believed to control the activity of proteins by regulating their folding, assembly, and intracellular trafficking (2–4). There are three families of PPIases, namely the cyclophilin (CyP), FK506-binding protein, and parvulin families. The CyP and FK506-binding protein families have been well established as targets of the immunosuppressants cyclosporin A and FK506, respectively (5–7).Together with Pin1, human parvulin (Par14, EPVH) constitutes the parvulin family and has been identified in all hitherto examined human tissues (8, 9). Par14 comprises 131 amino acid residues and has a 35-residue amino-terminal region that does not have sequence similarity to the WW domain (known to bind to phosphorylated serine/threonine-proline bonds in proteins and peptides) of Pin1. Phosphorylation at Ser-19 in this region regulates the subcellular localization and DNA binding activity of Par14; the phosphorylation is required for nuclear localization, and the dephosphorylation is a prerequisite for the binding of the first 25 residues to nuclear DNA (10). The 96-residue carboxyl-terminal domain has a 34.2% sequence identity with the PPIase domain of Pin1. Par14 reportedly has a substrate preference for positively charged residues preceding proline but not for phosphorylated Thr or Ser as is the case with Pin1; however, its rate constant for the prolyl cis to trans isomerization reaction is at least 1,000-fold lower than that of CyPs (9). NMR solution structural analysis has shown that Par14 folds into a βα3βαβ2 structure, which is essentially identical to that of Pin1 (11). The unstructured 35-residue amino-terminal region contains several basic residues and replaces the WW domain of Pin1 (11). This structural model explains the molecular basis for the preferential substrate specificity of Par14 for positively charged residues preceding proline as well as the putative role of the amino-terminal region as a DNA-binding domain. However, the physiological function of Par14 remains unknown.We previously reported that Par14 associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes as well as with many proteins that are implicated in the regulation of microtubule assembly or nucleolar reformation during mitosis (12, 13). We have proposed that Par14 is involved in ribosome biogenesis and/or nucleolar reassembly in mammalian cells during the pre- or postmitotic phases of the cell cycle. In the present study, we describe the comprehensive identification of protein components of the Par14-associated pre-rRNP complexes and establish Par14 as a de facto component of the pre-rRNP complexes in vivo. We also demonstrate that Par14 functions as a ribosomal RNA processing factor in mammalian ribosome biogenesis.     

Phylogenetic analyses of Japanese species of Phyllosticta sensu stricto

Although the genus concept of Phyllosticta s. str. (teleomorph: Guignardia) as defined by van der Aa is widely accepted, the species concept is still controversial because it is often based on the morphology on host plants. In this study, the culture characteristics within Phyllosticta s.str. were examined, and the phylogenetic relationships among Japanese species of Phyllosticta s.str. and its teleomorph Guignardia were analyzed using 18S rDNA sequences. Phyllosticta s. str. formed a monophyletic clade. ITS-28S rDNA sequences extracted from fungal cultures derived from various host plants were divided into two subgroups. The first group included cultures from a wide range of host plants and were mainly derived as endophytes from a symptom-less plant. In the second group, cultures from each host plant genus formed distinct clades; these were often isolated as leaf pathogens from diverse plants. Isolates belonging to the first lineage generally grew faster on oatmeal agar. To classify species of Phyllosticta it is necessary to consider an integrated approach such as molecular phylogeny, host plant, colony growth, symptoms, and morphological characteristics of the conidiomata.     

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide

Introduction

Immunosuppressants, including anti-TNFα antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis.

Methods

DBA/1 mice were immunized with hGPI_325-339, and cells of draining lymph node (DLN) were stimulated with hGPI_325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4⁺ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI_325-339 were synthesized. CD4⁺ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI_325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI_325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA.

Results

Human GPI_325-339-specific Th17 cells showed predominant usage of TCRVβ8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4⁺ T cells in the presence of hGPI_325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL.

Conclusions

We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.