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1.  Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1 
In this work, Pz peptidase B, an intracellular M3 metallopeptidase that is found in the thermophile Geobacillus collagenovorans MO-1, was crystallized using the counter-diffusion method.
Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as ‘Kibo’) at the International Space Station (ISS). The crystal belonged to the trigonal space group P3121, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.
PMCID: PMC3388914  PMID: 22750857
Pz peptidase B; Geobacillus collagenovorans MO-1; microgravity
2.  Survey of the Japanese Coast Reveals Abundant Placozoan Populations in the Northern Pacific Ocean 
Scientific Reports  2014;4:5356.
Placozoans are the simplest extant free-living animals consisting of only five cell types and lacking neurons and muscle cells. Their phylogenetic position implies they are important for uncovering the origins of metazoans. Although recent studies show multiple groups within the phylum, most placozoan research has been performed on laboratory-cultured clones deriving from a single specimen. Reports of placozoan discovery are concentrated in the tropic and subtropic seas, especially in the Mediterranean and the Caribbean. Here, I report the unexpected abundance of placozoans from the Japanese coast. They were found from all six studied sites, even during winter for two sites, suggesting that they are more tolerant to low temperatures than previously regarded. These results suggest an unknown abundance of placozoans in the Northern Pacific Ocean and further studies on these populations may be essential in solving important biological problems of the phylum.
PMCID: PMC4062898  PMID: 24942227
3.  Acoelomorph flatworms are deuterostomes related to Xenoturbella 
Nature  2011;470(7333):255-258.
Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data haverevised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes1,2 and positioning the Acoelomorpha as the most basally branching bilaterian group(s)3–6. Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria7. Here we assemble three independent data sets—mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements—and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements8 have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.
PMCID: PMC4025995  PMID: 21307940
4.  Two Types of Endosymbiotic Bacteria in the Enigmatic Marine Worm Xenoturbella bocki▿ †  
Two types of endosymbiotic bacteria were identified in the gastrodermis of the marine invertebrate Xenoturbella bocki (Xenoturbellida, Bilateria). While previously described Chlamydia-like endosymbionts were rare, Gammaproteobacteria distantly related to other endosymbionts and pathogens were abundant. The endosymbionts should be considered when interpreting the poorly understood ecology and evolution of Xenoturbella.
PMCID: PMC2849209  PMID: 20139320
5.  Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A from Geobacillus collagenovorans MO-1 
Pz peptidase A has been cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits thimet oligopeptidase and neurolysin.
Pz peptidase A is an intracellular M3 metallopeptidase found in the thermophile Geobacillus collagenovorans MO-1 that recognizes collagen-specific tripeptide units (Gly-Pro-Xaa). Pz peptidase A shares common reactions with mammalian thimet oligopeptidase (TOP) and neurolysin, but has extremely low primary sequence identity to these enzymes. In this work, Pz peptidase A was cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits TOP and neurolysin. The crystals belong to space group P21, with unit-cell parameters a = 56.38, b = 194.15, c = 59.93 Å, β = 106.22°. This is the first crystallographic study of an M3 family peptidase–PPI complex.
PMCID: PMC2330125  PMID: 17277461
Pz peptidase A; M3 metallopeptidases; collagen degradation; Geobacillus collangenovorans MO-1
6.  Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1 
Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.
PMCID: PMC2225360  PMID: 17142913
proline-specific aminopeptidase; Aneurinibacillus sp. strain AM-1; thermophiles
7.  Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3 
A plant- and prokaryote-conserved domain (PPC) has been crystallized. The crystal diffracted to 1.7 Å resolution and belonged to space group P6322.
A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 53.69, c = 159.2 Å. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 Å. A complete data set from crystals of the SeMet-substituted protein was also obtained.
PMCID: PMC1952417  PMID: 16511056
Pyrococcus horikoshii; PPC domain; AHL1
8.  Cysteinyl-tRNACys Formation in Methanocaldococcus jannaschii: the Mechanism Is Still Unknown 
Journal of Bacteriology  2004;186(1):8-14.
Most organisms form Cys-tRNACys, an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Söll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the “missing” CysRS activity for in vivo Cys-tRNACys formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the Km for cysteine and a ninefold reduction in the kcat for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNACys synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNACys formation in M. jannaschii still remains to be discovered.
PMCID: PMC303452  PMID: 14679218
9.  Xenoturbella bocki exhibits direct development with similarities to Acoelomorpha 
Nature Communications  2013;4:1537-.
Xenoturbella bocki, a marine animal with a simple body plan, has recently been suggested to be sister group to the Acoelomorpha, together forming the new phylum Xenacoelomorpha. The phylogenetic position of the phylum is still under debate, either as an early branching bilaterian or as a sister group to the Ambulacraria (hemichordates and echinoderms) within the deuterostomes. Although development has been described for several species of Acoelomorpha, little is known about the life cycle of Xenoturbella. Here we report the embryonic stages of Xenoturbella, and show that it is a direct developer without a feeding larval stage. This mode of development is similar to that of the acoelomorphs, supporting the newly proposed phylum Xenacoelomorpha and suggesting that the last common ancestor of the phylum might have been a direct developer.
Xenoturbella is a simple marine worm recently suggested to be either a deuterostome or an early branching bilaterian. Nakano et al. report the first observations of naturally spawned eggs and embryos from Xenoturbella, and uncover new insights into the evolutionary history of metazoan development.
PMCID: PMC3586728  PMID: 23443565
10.  Development of ciliary bands in larvae of the living isocrinid sea lily Metacrinus rotundus 
Embryos and larvae of an isocrinid sea lily, Metacrinus rotundus, are described by scanning electron microscopy. Around hatching (35 h after fertilization), the outer surface of the gastrula becomes ubiquitously covered with short cilia. At 40 h, the hatched swimming embryo develops a cilia-free zone of ectoderm on the ventral side. By 3 days, the very early dipleurula larva develops a cilia-free zone ventrally, densely ciliated regions laterally, and a sparsely ciliated region dorsally. At this stage, the posterior and anterior ciliary bands first appear: the former runs along a low ridge separating the densely from the sparsely ciliated epidermal regions, while the latter is visible, at first discontinuously, along the boundary between the densely ciliated lateral regions and the cilia-free ventral zone. In the late dipleurula larva (5 days after fertilization), the anterior and posterior loops of ciliary bands are well defined. The transition from the dipleurula to the semidoliolaria larva occurs at 6 days as the posterior loop becomes rearranged to form incompletely circumferential ciliary bands. The larva becomes competent to settle at this stage. The arrangement of the ciliary bands on the semidoliolaria is maintained during the second week of development, while the larva retains its competence to settle. The larval ciliary patterns described here are compared with those of stalkless crinoids and eleutherozoan echinoderms. The closest morphological similarities are between M. rotundus and the basal eleutherozoan class Asteroidea.
PMCID: PMC4302253  PMID: 25641974
sea lily; crinoid; isocrinid; echinoderm; Articulata; ciliary band; Metacrinus rotundus; dipleurula; doliolaria; scanning electron microscopy

Results 1-10 (10)