Shugo Watabe

Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.

Draft Genome of the Pearl Oyster Pinctada fucata: A Platform for Understanding Bivalve Biology

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry.

Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

Background Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. Principal Findings We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. Conclusions We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

Changes in carp myosin ATPase induced by temperature acclimation

SummaryMyosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca2+-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 μmol Pi·min-1·mg-1 at 0.2 M KCl for cold-acclimated carp and 0.47 μmol Pi·min-1·mg-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca2+-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 μmol Pi·min-1·mg-1 at pH 6 and 0.4 μmol Pi·min-1·mg-1 at pH 9. K+(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 μmol Pi·min-1·mg-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg2+-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 μmol Pi·min-1·mg-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg2+-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca2+-ATPase was 25·10-4·s-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.

STRUCTURE OF FUCOSE BRANCHES IN THE GLYCOSAMINOGLYCAN FROM THE BODY WALL OF THE SEA CUCUMBER STICHOPUS JAPONICUS

Fucose-branched chondroitin sulfate E was prepared from the body wall of sea cucumber Stichopus japonicus. The purified glycosaminoglycan (GAG) was chemically desulfated, followed by carboxyl reduction. Intact, desulfated, and desulfated/carboxyl-reduced GAG fractions were subjected to per-O-methylation. GC-MS analyses of the resultant partially methylated alditol acetates demonstrated that the fucose branch is formed by two fucopyranosyl residues linked glycosidically through position (1-->3), and that the fucose branch and glucuronic acid are almost equimolar. In addition, it was elucidated that about 20% of the branches stretch from O-3 position of a glucuronic acid moiety of the core chondroitin sulfate polymer, while remaining fucose branches are postulated to protrude from O-4 and/or O-6 position(s) of a N-acetylgalactosamine moiety. This fucose branch was also confirmed to be highly sulfated according to six kinds of substitution pattern in methylation analysis.

Toughness and Collagen Content of Abalone Muscles

Toughness and collagen content were measured for various muscle parts of the Japanese abalone, kuro-awabi (Haliotis discus), in relation to muscle structures. The dorsal surface of the foot was toughest, followed by the hard and soft part of the foot, then the upper and middle part of the adductor muscle, irrespective of being reared or wild specimens. When compared with other abalone species, kuro-awabi showed the highest toughness for all the muscle parts, followed by madaka (H. sieboldii) and megai-awabi (H. gigas), while ezo-awabi (H. discus hannai) was softest. Collagen content was parallel with muscle toughness: the higher the collagen content, the tougher the muscle. Light micrographs of kuro-awabi showed that foot and the dorsal surface of foot were dominated by connective tissues, while adductor muscle was mainly composed of myofibrils. Transmission electron micrographs demonstrated that myofibrils in the foot were surrounded by thick layers of collagen fibrils of about 1 μm, confirming light microscopic observations.

Phosphorylation of a high molecular weight (∼600 kDa) protein regulates catch in invertebrate smooth muscle

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called ‘catch’. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of ∼600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the ∼600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of ∼600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the ∼600 kDa protein regulates catch

Dual Roles of Notch in Regulation of Apically Restricted Mitosis and Apicobasal Polarity of Neuroepithelial Cells

How the mitosis of neuroepithelial stem cells is restricted to the apical ventricular area remains unclear. In zebrafish, the mosaic eyes(rw306) (moe/epb41l5(rw306)) mutation disrupts the interaction between the putative adaptor protein Moe and the apicobasal polarity regulator Crumbs (Crb), and impairs the maintenance of neuroepithelial apicobasal polarity. While Crb interacts directly with Notch and inhibits its activity, Moe reverses this inhibition. In the moe(rw306) hindbrain, Notch activity is significantly reduced, and the number of cells that proliferate basally away from the apical area is increased. Surprisingly, activation of Notch in the moe(rw306) mutant rescues not only the basally localized proliferation but also the aberrant neuroepithelial apicobasal polarity. We present evidence that the Crb⋅Moe complex and Notch play key roles in a positive feedback loop to maintain the apicobasal polarity and the apical-high basal-low gradient of Notch activity in neuroepithelial cells, both of which are essential for their apically restricted mitosis.

Paramyosin and the catch mechanism

1. Catch is a mechanism found in many molluscan smooth muscles in which tension is maintained at relatively low energy cost. 2. Paramyosin forms the core of thick filaments. In catch muscle paramyosin concentrations are high and the thick filaments are relatively long. 3. The mechanism of catch is not understood, but the consensus is that tension during catch is borne by slowly-cycling cross-bridge attachments to actin. 4. Stimulation by acetylcholine increases intracellular Ca2+ and initiates a contraction characterized by a relatively rapid cross-bridge cycling. Reduction of Ca2+ can lead to relaxation or catch. Relaxation occurs only when a second neurotransmitter, serotonin, is present. 5. The catch state is released by serotonin, via activation of adenylate cyclase, increased levels of cAMP and phosphorylation of one or more contractile proteins, possibly paramyosin. Other targets for phosphorylation are discussed. 6. The contractile cycle of catch muscles, therefore, is controlled by both Ca2+ and cAMP.

Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch

SUMMARY Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini, respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.