Biomedical subjects

Takashi Morita

Publications and source records attributed to Takashi Morita.

At least 19 recordsLinked to original sources

PubMed2006

Mitochondria-related male infertility.

Approximately 15% of human couples are affected by infertility, and about half of these cases of infertility can be attributed to men, through low sperm motility (asthenozoospermia) or/and numbers (oligospermia). Because mitochondrial genome (mtDNA) mutations are identified in patients with fertility problems, there is a possibility that mitochondrial respiration defects contribute to male infertility. To address this possibility, we used a transmitochondrial mouse model (mito-mice) carrying wild-type mtDNA and mutant mtDNA with a pathogenic 4,696-bp deletion (DeltamtDNA). Here we show that mitochondrial respiration defects caused by the accumulation of DeltamtDNA induced oligospermia and asthenozoospermia in the mito-mice. Most sperm from the infertile mito-mice had abnormalities in the middle piece and nucleus. Testes of the infertile mito-mice showed meiotic arrest at the zygotene stage as well as enhanced apoptosis. Thus, our in vivo study using mito-mice directly demonstrates that normal mitochondrial respiration is required for mammalian spermatogenesis, and its defects resulting from accumulated mutant mtDNAs cause male infertility.

Animals↗

PubMed2006

Localization of heparin- and neuropilin-1-recognition sites of viral VEGFs.

VEGF-A165 plays a central role in neovascularization. The biological activities of VEGF-A165 are largely mediated through KDR. VEGF-A165 also binds to cellular coreceptors, neuropilin-1 (NP-1), and heparin, via its C-terminal domain, resulting in functional modulation. Parapoxvirus-encoded VEGFs (PV-VEGFs), which recognize KDR, possess basic amino acid clusters in their C-terminal regions. Some PV-VEGFs may interact with NP-1; however, the NP-1- and heparin-binding properties have not been fully characterized. Here, we demonstrate that the heparin- and NP-1-binding region of PV-VEGFs is located in its C-terminal tail. Furthermore, the two arginine residues adjacent to the C-terminus greatly contribute to both interactions.

Binding Sites↗

PubMed2006

The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells.

A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.

Brain↗

PubMed2006

Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX.

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.

Amino Acid Sequence↗

PubMed2006

Molecular and functional diversity of vascular endothelial growth factors.

Members of the vascular endothelial growth factor (VEGF) family are crucial regulators of neovascularization and are classified as cystine knot growth factors that specifically bind cellular receptor tyrosine kinases VEGFR-1, VEGFR-2, and VEGFR-3 with high but variable affinity and selectivity. The VEGF family has recently been expanded and currently comprises seven members: VEGF-A, VEGF-B, placenta growth factor (PlGF), VEGF-C, VEGF-D, viral VEGF (also known as VEGF-E), and snake venom VEGF (also known as VEGF-F). Although all members are structurally homologous, there is molecular diversity among the subtypes, and several isoforms, such as VEGF-A, VEGF-B, and PlGF, are generated by alternative exon splicing. These splicing isoforms exhibit differing properties, particularly in binding to co-receptor neuropilins and heparin. VEGF family proteins play multiple physiological roles, such as angiogenesis and lymphangiogenesis, while exogenous members (viral and snake venom VEGFs) display activities that are unique in physiology and function. This review will highlight the molecular and functional diversity of VEGF family proteins.

Amino Acid Sequence↗

PubMed2006

Atorvastatin therapy associated with improvement in left ventricular remodeling in a case of idiopathic dilated cardiomyopathy.

Statins have pleiotropic effects such as anti-inflammatory and vascular protective effects that would be beneficial for patients with chronic heart failure. This report describes a patient with idiopathic dilated cardiomyopathy and a long-standing history of heart failure that was treated with atorvastatin in addition to conventional therapy that included beta-blockers. Atorvastatin therapy for 12 months was associated with an improvement in cardiac function and improved left ventricular remodeling and peak oxygen consumption. This result suggests that statin therapy may be a potential novel treatment strategy for patients with chronic heart failure.

Adult↗

PubMed2006

Different contributions of polymorphisms in VKORC1 and CYP2C9 to intra- and inter-population differences in maintenance dose of warfarin in Japanese, Caucasians and African-Americans.

OBJECTIVE: To investigate pharmacokinetic and pharmacodynamic factors associated with population differences in warfarin doses needed to achieve anticoagulation, in particular the possible involvement of genetic variability in vitamin K epoxide reductase (VKOR) and CYP2C9. METHODS: Warfarin maintenance dose, unbound plasma S-warfarin concentration [Cu(S)] and INR were determined in 157 Caucasians, 172 Japanese, and 36 African-Americans stably anticoagulated patients. In a subset (n = 166), fully carboxylated plasma normal prothrombin levels (NPT) were also measured. Genotyping for seven CYP2C9 (CYP2C9*1 through 6 and *11) and seven VKORC1 variants were performed in 115 Caucasians and 64 Japanese patients and 66 healthy African-Americans. Multivariate analysis was performed to identify covariates associated with warfarin requirement. RESULTS: The relationship between NPT and Cu(S) indicated Japanese are more susceptible to inhibition of NPT production by S-warfarin than the other two populations. VKORC1 1173 C > T had a greater frequency in Japanese (89.1%) than Caucasians (42.2%) and African-Americans (8.6%). CYP2C9 variants with reduced metabolizing ability were less frequent in Japanese compared to the other two populations. The median warfarin dose was significantly higher in Caucasians than Japanese patients (5.5 versus 3.5 mg/day), however, when matched for CYP2C9*1 homozygosity, no difference in dose was observed between VKORC1 genotype-matched groups. Furthermore, VKORC1 1173C > T and CYP2C9 (*2/*3/*11) genotypes, age and weight were identified as independent covariates contributing to interpatient variability in warfarin dosage. CONCLUSIONS: Both VKORC1 and CYP2C9 polymorphisms contribute to inter-population difference in warfarin doses among the three populations, but their contribution to intra-population variability may differ within each population.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

pH-Dependent structural changes at Ca(2+)-binding sites of coagulation factor IX-binding protein.

Coagulation factor IX-binding protein, isolated from Trimeresurus flavoviridis (IX-bp), is a C-type lectin-like protein. It is an anticoagulant consisting of homologous subunits, A and B. Each subunit has a Ca(2+)-binding site with a unique affinity (K(d) values of 14muM and 130muM at pH 7.5). These binding characteristics are pH-dependent and, under acidic conditions, the Ca(2+) binding of the low-affinity site was reduced considerably. In order to identify which site has high affinity and to investigate the pH-dependent Ca(2+) release mechanism, we have determined the crystal structures of IX-bp at pH 6.5 and pH 4.6 (apo form), and compared the Ca(2+)-binding sites with each other and with those of the solved structures under alkaline conditions; pH 7.8 and pH 8.0 (complexed form). At pH 6.5, Glu43 in the Ca(2+)-binding site of subunit A displayed two conformations. One (minor) is that in the alkaline state, and the other (major) is that at pH 4.6. However, the corresponding Gln43 residue of subunit B is in only a single conformation, which is almost identical with that in the alkaline state. At pH 4.6, Glu43 of subunit A adopts a conformation similar to that of the major conformer observed at pH 6.5, while Gln43 of subunit B assumes a new conformation, and both Ca(2+) positions are occupied by water molecules. These results showed that Glu43 of subunit A is much more sensitive to protonation than Gln43 of subunit B, and the conformational change of Glu43 occurs around pH6.5, which may correspond to the step of Ca(2+) release.

Animals↗

PubMed2005

A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2.

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Crystal structure of a CRISP family Ca2+ -channel blocker derived from snake venom.

The cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals, reptiles, amphibians and secernenteas, and are involved in a variety of biological reactions. Here we report the crystal structure of triflin, a snake venom derived blocker of high K(+)-induced artery contraction, at 2.4A resolution. Triflin consists of two domains. The first 163 residues form a large globular body with an alpha-beta-alpha sandwich core, which resembles pathogenesis-related proteins of group-1 (PR-1). Two glutamic acid-associated histidine residues are located in an elongated cleft. A Cd(2+) resides in this binding site, and forms a five-coordination sphere. The subsequent cysteine-rich domain adopts a rod-like shape, which is stabilized by five disulfide bridges. Hydrophobic residues, which may obstruct the target ion-channel, are exposed to the solvent. A concave surface, which is surrounded by these two domains, is also expected to play a significant role in the binding to the target receptor, leading to ion channel blockage. The C-terminal cysteine-rich region has a similar tertiary structure to voltage-gated potassium channel blocker toxins, such as BgK and ShK. These findings will contribute toward understanding the functions of the widely distributed CRISP family proteins.

Animals↗

PubMed2005

Specific distribution of VEGF-F in Viperinae snake venoms: isolation and characterization of a VGEF-F from the venom of Daboia russelli siamensis.

Vascular endothelial growth factor exerts multiple functions through binding to two distinct receptor tyrosine kinases, Flt-1 and KDR. We previously demonstrated that snake venom VEGFs, designated VEGF-Fs, exhibit potent biological activities when compared with VEGF165 and that they selectively recognize KDR. We herein report the screening of several snake venoms for VEGF-Fs using antibodies against vammin and HF, VEGF-Fs derived from the venom of Vipera ammodytes ammodytes and Vipera aspis aspis, respectively. Specific immunoreactivity was observed in the venoms of Vipera and Daboia species. The present results suggest that VEGF-Fs are specific components of venoms from the Viperinae snakes that inhabit Europe, India, and Asia. We also report the isolation and characterization of an additional VEGF-F from Daboia russelli siamensis venom.

Amino Acid Sequence↗

PubMed2005

Identification of vascular endothelial growth factor receptor-binding protein in the venom of eastern cottonmouth. A new role of snake venom myotoxic Lys49-phospholipase A2.

Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.

Agkistrodon↗

PubMed2005

Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 A, and diffraction data were collected to 2.25 A resolution. Crystals of Pdc also belonged to space group P2(1)2(1)2(1), with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 A, and diffraction data were collected to 1.90 A resolution.

Animals↗

PubMed2005

Identification of the heparin-binding region of snake venom vascular endothelial growth factor (VEGF-F) and its blocking of VEGF-A165.

VEGF-A165 displays multiple effects through binding to KDR (VEGFR-2). Heparin/heparan sulfate-like molecules are known to greatly modulate their interaction. In fact, VEGF-A lacking a C-terminal heparin-binding region exhibits significantly reduced mitogenic activity. We recently found novel heparin-binding VEGFs in snake venom, designated VEGF-Fs, which specifically recognize KDR, rather than other VEGF receptors. VEGF-Fs virtually lack the C-terminal heparin-binding region when compared with other heparin-binding VEGF subtypes, despite their heparin-binding potential. The C-terminal region does not exhibit any significant homology with other known proteins or domains. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. The C-terminal peptide of vammin (one of the VEGF-Fs, 19 residues) bound to heparin with similar affinity as native vammin. We then evaluated the effects of this peptide on the biological activity of VEGF-A165. The C-terminal peptide of VEGF-F exhibited specific blockage of VEGF-A165 activity both in vitro and in vivo. These observations demonstrate that the short C-terminal region of VEGF-F functions fully as the active heparin-binding domain and the corresponding peptide specifically blocks VEGF-A165, thus suggesting that the C-terminal heparin-binding region of VEGF-F recognizes similar heparin/heparan sulfate molecules as VEGF-A165. The present results will provide novel insight into VEGF-heparin interaction and may facilitate the design of new anti-VEGF drugs based on novel strategies.

Amino Acid Sequence↗

PubMed2005

RNA interference during spermatogenesis in mice.

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.

Animals↗

PubMed2005

Structures and functions of snake venom CLPs (C-type lectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities.

C-type lectin-like proteins (CLPs) have a variety of biological activities, including anticoagulant- and platelet-modulating activities but have no lectin activity. CLPs are made up of heterodimers or oligomers of heterodimers, while C-type lectins from snake venom are composed exclusively of homodimers or homooligomers. In the last decade, numerous CLPs, such as blood coagulation factor IX/X-binding protein and botrocetin, have been isolated from various snake venoms, sequenced, and characterized. In addition, RVV-X (factor X activator) and carinactivase-1 (prothrombin activator) are metalloproteases composed of two C-type lectin-like domains that recognize the Gla domain of factor X and prothrombin, respectively. The basic structures of these CLPs include two homologous subunits: subunit alpha (A chain) of 14-15 kDa and subunit beta (B chain) of 13-14 kDa. CLPs occur in a variety of oligomeric forms, including alphabeta, (alphabeta)(2), and (alphabeta)(4). The basic homologous dimer (alphabeta) of these CLPs is formed by three-dimensional (3D) domain swapping. The CLPs constitute a new protein family and are useful tools for elucidating the mechanisms involved in clotting and platelet activation as well as the structure-function relationships of both blood clotting factors and platelet glycoproteins.

Amino Acid Sequence↗

PubMed2005

Structure-function relationships of C-type lectin-related proteins.

The structural and functional studies of the first identified C-type lectin-like protein (CLP), blood coagulation factor IX/factor X-binding protein (IX/X-bp), have been instrumental in defining how new functionally heterodimeric CLPs are generated from monomeric carbohydrate recognition domain in C-type lectins by three-dimensional domain swapping. The crystal structures of gamma-carboxyglutamic acid domains of coagulation factors X and IX have recently been clarified in structural studies of complexes between the gamma-carboxyglutamic acid domain of factors X and X-bp (a venom CLP) and between the gamma-carboxyglutamic acid domain of factors IX and IX-bp (a venom CLP).

1-Carboxyglutamic Acid↗

PubMed2005

Identification and localization of heparin-binding region of snake venom VEGF and its blocking of VEGF-A165.

Vascular endothelial growth factor (VEGF-A165) displays diverse effects through binding to its receptor, KDR (kinase domain-containing receptor). Heparan sulfate/heparin-like molecules greatly contribute to their interaction. Indeed, the mitogenic potency of VEGF-A165 lacking the C-terminal heparin-binding region is less than 1% compared with intact VEGF-A165. We previously found novel heparin-binding VEGFs, designated VEGF-F that specifically recognizes KDR in snake venoms. VEGF-Fs almost completely lack the C-terminal heparin-binding region compared with VEGF-A165, despite their heparin-binding potential. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. We have demonstrated that the heparin-binding site of VEGF-F is located in its C-terminal region, particularly localized on the N-terminal portion of this region. Furthermore, a synthetic peptide of this region blocks the biological activity of VEGF-A165 in vitro and in vivo.

Animals↗