Biomedical subjects

Kazuo Inaba

Publications and source records attributed to Kazuo Inaba.

At least 19 recordsLinked to original sources

PubMed2006

A novel oocyte maturation arresting factor in the central nervous system of scallops inhibits serotonin-induced oocyte maturation and spawning of bivalve mollusks.

Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph. Histological observation demonstrated that the novel inhibitor prevented 5-HT from inducing oocyte maturation in the scallop ovary and that no prostaglandin F2alpha (PGF2alpha) inhibited 5-HT-induced oocyte maturation, although PGF2alpha strongly prohibited 5-HT-induced egg release through the gonoduct from ovarian tissue. The novel inhibitor from the scallop CPG also prohibited 5-HT-induced oocyte maturation of other bivalve species as well as scallops. The novel inhibitor, mediated through a receptor mechanism on oocyte membranes, blocked extracellular Ca2+ uptake into oocytes, which was observed in 5-HT-induced oocyte maturation. It is suggested that the novel inhibitor with a molecular mass of 60 kDa, named oocyte maturation arresting factor, which appears to be a universal substance for bivalve species, may be transported from the CPG to the ovary via hemolymph and may prohibit 5-HT-induced oocyte maturation due to the interference of extracellular Ca2+ influx into oocytes, eventually resulting in the inhibition of spawning. On the other hand, it seems that PGF2alpha inhibits 5-HT-induced transport of mature eggs through the gonoduct.

Animals↗

PubMed2006

Ap58: a novel in situ outer dynein arm-binding protein.

Outer arm dynein is a molecular motor that is positioned at 24 nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58 kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58 kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein by a high salt solution from axonemes. Sucrose density gradient centrifugation or gel filtration of the extract separates the outer arm dynein core from ap58. Most ap58 sediments to the lower density fraction or elutes in fractions of smaller molecules. However, immunogold localization reveals that ap58 is distributed at approximately 25 nm intervals on doublet microtubules, suggesting that in situ it is associated with the outer dynein arm. Thus, ap58 with the TPR motif is a new member of outer dynein arm-binding proteins distinct from the outer dynein arm-docking complex.

Amino Acid Sequence↗

PubMed2006

Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2.

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.

Animals↗

PubMed2006

Association of a 66 kDa homolog of Chlamydomonas DC2, a subunit of the outer arm docking complex, with outer arm dynein of sperm flagella in the ascidian Ciona intestinalis.

We previously identified a 66 kDa axonemal protein (Ci-Axp66.0) in sperm of the ascidian Ciona intestinalis. Here we found that Ci-Axp66.0 shows sequence similarity to the DC2 subunit of the Chlamydomonas outer arm docking complex. Analysis of secondary structure of Ci-Axp66.0 suggested that the N-terminal two-thirds of the molecule is rich in coiled coil structure, as in Chlamydomonas DC2. Immunogold localization revealed that it is located in the vicinity of outer arm dynein. Ci-Axp66.0 was partly extracted from the axonemes by a high salt solution and co-purified with outer arm dynein. This co-purification was not affected by the absence of Mg(2+) in isolation buffer, indicating that Ci-Axp66.0 is associated with outer arm dynein. These results suggest that Ci-Axp66.0 is a component of the outer arm dynein docking complex in the axonemes of Ciona sperm.

Amino Acid Sequence↗

PubMed2005

Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.

Changes in membrane potential affect ion channels and transporters, which then alter intracellular chemical conditions. Other signalling pathways coupled to membrane potential have been suggested but their underlying mechanisms are unknown. Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1-S4 segments of voltage-gated channels and a cytoplasmic domain similar to phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing phosphatase (Ci-VSP), displays channel-like 'gating' currents and directly translates changes in membrane potential into the turnover of phosphoinositides. The activity of the phosphoinositide phosphatase in Ci-VSP is tuned within a physiological range of membrane potential. Immunocytochemical studies show that Ci-VSP is expressed in Ciona sperm tail membranes, indicating a possible role in sperm function or morphology. Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized.

Amino Acid Sequence↗

PubMed2004

Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian Ciona intestinalis.

Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with radial spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of radial spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk, possibly at the junction between spoke head and the stalk.

Animals↗

PubMed2004

Expression of 17beta-hydroxysteroid dehydrogenease in testis of the ascidian Ciona intestinalis [corrected].

Ascidians have been employed as model organisms in investigating spermatogenesis. 17beta-hydroxysteroid dehydrogenase (HSD) is a steroidogenic enzyme essential for invertebrate spermatogenesis. A homologue of HSD was found in the EST database of Ciona intestinalis and cloned. Sequence analysis showed significant homology to zebra fish, sea urchin and human 17beta-HSD. The gene has an open reading frame (ORF) of 918 nucleotides coding for a polypeptide of 306 amino acids and a calculated mass of 35-kDa. Immunoblotting with an antibody raised against HSD recognized a 35-kDa protein purified from the C. intestinalis testis. The HSD protein was localized in steroidogenic cells in the Ciona testis. These results suggest that C. intestinalis 17beta-HSD is equivalent to the enzyme of vertebrate Leydig cells and that 17beta-HSD could be a phylogenetic marker for organisms producing steroids.

17-Hydroxysteroid Dehydrogenases↗

PubMed2004

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis.

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.

Animals↗

PubMed2003

Sperm motility-activating complex formed by t-complex distorters.

Transmission ratio distortion is a dramatic example of non-Mendelian transmission. In mice, t-haplotype males produce dysfunctional +-sperm and normal t-sperm, leading to transmission in favor of t-sperm. Genetic studies have indicated that the t-complex responder locus, Tcr, rescues t-sperm but not +-sperm from defective products of t-complex distorter loci, Tcds. Light chain 1 (LC1) and LC3 from sea urchin sperm outer arm dynein have sequence similarities to Tctex2 and Tctex1, respectively, both of which are wild-type products of Tcds. We show here that LC1 and LC3 are able to make a 1:1 complex. Since Tcr is a member of the Smok (sperm motility kinase) family and LC1 is phosphorylated at the activation of sperm motility in a cAMP-dependent manner, this complex in a dynein motor molecule might be a direct target of Smok/Tcr kinase in a signal cascade that regulates sperm motility. Thus, we designate it as Smoac (sperm motility activating complex).

Amino Acid Sequence↗

PubMed2003

DC3, the 21-kDa subunit of the outer dynein arm-docking complex (ODA-DC), is a novel EF-hand protein important for assembly of both the outer arm and the ODA-DC.

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme (Takada et al. 2002. Mol. Biol. Cell 13, 1015-1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.

Amino Acid Sequence↗

PubMed2003

Characterization of a cAMP-dependent protein kinase catalytic subunit from rainbow trout spermatozoa.

The cyclic AMP-dependent phosphorylation of proteins is essential for the initiation of sperm motility in salmonid fishes. This study isolated cDNA for the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) from rainbow trout testis. The deduced amino acid sequence shows 75-80% identity to sequences previously reported in other organisms. However, the N-terminal regions of PKA-C from the testis as well as ovary in the trout appear slightly shorter than those from other tissues, suggesting that small PKA-C might be specific to germ cells. An immunofluorescence study using polyclonal antibody against trout testis PKA-C shows that it localizes along sperm flagellum. Furthermore, immunoelectron microscopy revealed that PKA-C is anchored to the outer arm dynein of flagellar axonemes. These results suggest that PKA-C is involved in regulating the flagellar motility of sperm via phosphorylation of a subunit of the outer arm dynein.

Amino Acid Sequence↗

PubMed2003

Control of flatfish sperm motility by CO2 and carbonic anhydrase.

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.

Animals↗

PubMed2003

Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis-initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility.

Exposure to hypo-osmotic or hyperosmotic environment triggers the initiation of fish sperm motility. In this article, we report that calcium and potassium channel blockers do not influence motility of puffer fish sperm but calmodulin antagonists reversibly decrease it, suggesting that calmodulin-Ca(2+) interactions are prerequisite for the initiation of sperm motility in this species. Gadolinium (a stretch activated ion channel blocker) decreased the motility of puffer fish sperm from 92 +/- 3% to 6 +/- 3% and that of carp sperm from 91 +/- 7% to 3.5 +/- 4.3% in a dose-dependent manner (10-40 micro M). The effect of gadolinium was reversible, suggesting that stretch activated ion channels participate in the initiation of sperm motility of the two species. Gadolinium inhibits changes in the isoelectric point of certain proteins of puffer fish sperm, which occur when sperm motility is initiated in a hypertonic solution. Anisotropy measurements showed that hypo-osmotic treatment, which initiates carp sperm motility, increased membrane fluidity. When hypo-osmotic treatment was given in the presence of gadolinium, the sperm membrane remained as rigid as in quiescent cells, while motility was blocked. By contrast, gadolinium did not influence the motility parameters of Ciona or human sperm. Based on these lines of evidence, we suggest that conformational changes of mechanosensitive membrane proteins are involved in osmolality-dependent but not osmolality-independent sperm.

Animals↗

PubMed2003

Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the Ascidian Ciona intestinalis.

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.

Amino Acid Sequence↗

PubMed2003

Molecular architecture of the sperm flagella: molecules for motility and signaling.

Sperm motility is generated by a highly organized, microtubule-based structure, called the axoneme, which is constructed from approximately 250 proteins. Recent studies have revealed the molecular structures and functions of a number of axonemal components, including the motor molecules, the dyneins, and regulatory substructures, such as radial spoke, central pair, and other accessory structures. The force for flagellar movement is exerted by the sliding of outer-doublet microtubules driven by the molecular motors, the dyneins. Dynein activity is regulated by the radial spoke/central pair apparatus through protein phosphorylation, resulting in flagellar bend propagation. Prior to fertilization, sperm exhibit dramatic motility changes, such as initiation and activation of motility and chemotaxis toward the egg. These changes are triggered by changes in the extracellular ionic environment and substances released from the female reproductive tract or egg. After reception of these extracellular signals by specific ion channels or receptors in the sperm cells, intracellular signals are switched on through tyrosine protein phosphorylation, Ca2+, and cyclic nucleotide-dependent pathways. All these signaling molecules are closely arranged in each sperm flagellum, leading to efficient activation of motility.

Animals↗

PubMed2002

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Alleles↗

PubMed2002

A chemoattractant for ascidian spermatozoa is a sulfated steroid.

Sperm chemotaxis toward eggs before fertilization has been demonstrated in many animals and plants, and several peptides and small organic compounds acting as chemoattractants have been identified. We previously showed that sperm of the ascidians Ciona intestinalis and Ciona savignyi are activated and then attracted toward the egg by a common factor released from the egg. In this study, we purified sperm-activating and -attracting factor (SAAF) from the egg-conditioning medium of C. intestinalis by using several steps of column chromatography. Determination of the molecular structure by NMR and MS/MS analysis revealed that SAAF is a previously uncharacterized sulfated steroid: 3,4,7,26-tetrahydroxycholestane-3,26-disulfate. Furthermore, it was shown that the SAAF of C. savignyi was indistinguishable from that of C. intestinalis in terms of the chromatographic behavior and molecular weight, indicating that the same compound might be responsible for sperm activation and chemotaxis in both the species. Furthermore, we established a method for quantitative analysis of sperm chemotaxis and showed that the chemotactic behavior of Ciona sperm is controlled by the "chemotactic turn" associated with decrease in the concentration of SAAF.

Animals↗

PubMed2002

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase.

Sperm flagellar movements are regulated by cAMP-dependent protein phosphorylation. Tctex2-related light chain of outer arm dynein is a well-defined phosphorylated protein that is phosphorylated at activation of sperm motility. Here, the protein phosphatase that dephosphorylates Tctex2-related dynein light chain (LC2) has been characterized in salmonid fish sperm. Most of the phosphatase activity against LC2 is found in Triton-soluble fraction of flagella but trace extent of the activity is retained in the axoneme. The dephosphorylation of LC2 is inhibited by okadaic acid at more than 1nM, whereas that of dynein alpha heavy chain is inhibited at more than 10nM. The addition of Ca(2+) gives no direct effect on LC2 dephosphorylation, but it accelerates the dephosphorylation of the regulatory subunit of cAMP-dependent protein kinase, resulting in the decrease of LC2 phosphorylation. The activity to dephosphorylate the LC2 is separated by MonoQ ion-exchange column chromatography along with the immunoreactivity to the antibody against the catalytic subunit of type 2A protein phosphatase. These results suggest that LC2 is dephosphorylated by type 2A protein phosphatase and that dynein alpha heavy chain and the regulatory subunit of cAMP-dependent protein kinase are dephosphorylated by other types of protein phosphatases.

Animals↗