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Biomedical subjects

K Mochida

Publications and source records attributed to K Mochida.

At least 37 records · Page 2Linked to original sources
PubMed2000

Development of embryos in superovulated guinea pigs following active immunization against the inhibin alpha-subunit.

Embryo recovery and subsequent embryonic development from guinea pigs treated with or without inhibin vaccines were compared to determine the effect of active immunization against the inhibin alpha-subunit. Twenty female guinea pigs of the Hartley strain were injected 3 times either with 1 ml inhibin vaccine (recombinant ovine inhibin a-subunit in oil emulsion: 50 microg/ml, inhibin-immunized group), or 1 ml placebo (saline in oil emulsion; control group) at 4 week intervals. After one estrous cycle following the last injection, females were naturally mated and embryos were collected at 11:00 hr of day 6 of pregnancy (Day 1: sperm in the vaginal smear) for culture in vitro. Active immunization increased the number of corpora lutea (12.6+/-3.0 vs. 4.6+/-0.2, P<0.05), recovered embryos (9.8+/-1.9 vs. 3.6+/-0.4, P<0.01) and normal embryos (7.8+/-1.4 vs. 3.6+/-0.4, P<0.05), although estrous cycle length was not affected (P>0.05). During subsequent 8 day culture in vitro, most of the recovered embryos formed trophoblast outgrowth; 100% (14/14) and 88.2% (15/17) in control and immunized groups, respectively. High levels of inhibin antibody titers were sustained in the inhibin-immunized guinea pigs at least for 5 months after the last injection while no antibody titer was detected in the control animals. These results indicate that active immunization against the inhibin a-subunit is a long-acting and efficient method to induce superovulation with normal embryonic development in the guinea pig.

Animals↗
PubMed1999

Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis.

A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response.

Apoptosis↗
PubMed1999

Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer.

The nodal gene is expressed on the left side of developing mouse embryos and is implicated in left/right (L-R) axis formation. The transcriptional regulatory regions of nodal have now been investigated by transgenic analysis. A node-specific enhancer was detected in the upstream region (-9.5 to -8.7 kb) of the gene. Intron 1 was also shown to contain a left side-specific enhancer (ASE) that was able to direct transgene expression in the lateral plate mesoderm and prospective floor plate on the left side. A 3. 5-kb region of nodal that contained ASE responded to mutations in iv, inv, and lefty-1, all genes that act upstream of nodal. The same 3. 5- kb region also directed expression in the epiblast and visceral endoderm at earlier stages of development. Characterization of deletion constructs delineated ASE to a 340-bp region that was both essential and sufficient for asymmetric expression of nodal. Several sequence motifs were found to be conserved between the nodal ASE and the lefty-2 ASE, some of which appeared to be essential for nodal ASE activity. These results suggest that similar transcriptional mechanisms underlie the asymmetric expression of nodal and of lefty-2 as well as the earlier expression of nodal in the epiblast and endoderm.

Animals↗
PubMed1999

Distinct transcriptional regulatory mechanisms underlie left-right asymmetric expression of lefty-1 and lefty-2.

Both lefty-1 and lefty-2 genes are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) axis formation. With the use of transgenic analysis, the transcriptional regulatory regions of these genes responsible for their L-R asymmetric expression have now been investigated. The 9.5-kb upstream region of lefty-1 and the 5.5-kb upstream region of lefty-2 reproduced the expression pattern of the corresponding gene. Examination of deletion constructs revealed the presence of a left side-specific enhancer (ASE) that is essential and sufficient for lefty-2 asymmetric expression. In contrast, the asymmetric expression of lefty-1 was shown to be determined by a combination of bilateral enhancers and a right side-specific silencer (RSS). The 9. 5-kb region of lefty-1 and the 5.5-kb region of lefty-2 responded to iv and inv, upstream genes of lefty-1 and lefty-2. The regulation of lefty-2 by iv and inv was mediated by ASE. These results suggest that, in spite of the similarities between lefty-1 and lefty-2, different regulatory mechanisms underlie their asymmetric expression.

Animals↗
PubMed1999

Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the azh/azh mutant mouse.

The tubulin-containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule-associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotype-specific tubulin antibodies as immunoprobes. Manchettes from wild-type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage-specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild-type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, alpha-3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild-type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase-contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso-like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation.

Animals↗
PubMed1999

GFR alpha3, a component of the artemin receptor, is required for migration and survival of the superior cervical ganglion.

GFR alpha3 is a component of the receptor for the neurotrophic factor artemin. The role of GFR alpha3 in nervous system development was examined by generating mice in which the Gfr alpha3 gene was disrupted. The Gfr alpha3-/- mice exhibited severe defects in the superior cervical ganglion (SCG), whereas other ganglia appeared normal. SCG precursor cells in the mutant embryos failed to migrate to the correct position, and they subsequently failed to innervate the target organs. In wild-type embryos, Gfr alpha3 was expressed in migrating SCG precursors, and artemin was expressed in and near the SCG. After birth, SCG neurons in the mutant mice underwent progressive cell death. These observations suggest that GFR alpha3-mediated signaling is required both for the rostral migration of SCG precursors and for the survival of mature SCG neurons.

Animals↗
PubMed1999

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

Activin Receptors, Type II↗
PubMed1999

A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, Oreochromis niloticus.

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT-30) against a molecular weight (Mr) = 120,000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration-dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT-30, suggesting that the TAT-30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with TAT-30 demonstrated that the sperm immobilizing factor was more than Mr = 1,000,000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120,000-TAT-30 positive protein. Additionally, lectin blot analysis showed that the TAT-30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT-30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells.

Animals↗
PubMed1999

Salmonid olfactory system-specific protein (N24) exhibits glutathione S-transferase class pi-like structure.

A salmonid olfactory system-specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as approximately 43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S-transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity-purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.

Animals↗
PubMed1999

Inhibitor studies of a new antibiotic, korormicin, 2-n-heptyl-4-hydroxyquinoline N-oxide and Ag+ toward the Na+-translocating NADH-quinone reductase from the marine Vibrio alginolyticus.

A new antibiotic, korormicin, isolated from a marine bacterium Pseudoalteromonas sp. F-420, was found to strongly inhibit the respiratory chain-linked Na+-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus. Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin specifically inhibited the Na+-dependent reaction in the NQR complex that is directly coupled to the extrusion of Na+ from the cells. Both korormicin and HQNO acted as purely noncompetitive inhibitors with regard to Q-1, and the inhibitor constants were estimated to be 82 pM and 0.3 microM, respectively. Mutual exclusiveness of korormicin and HQNO was analyzed by kinetic methods, which indicated that a part of the binding site of korormicin and HQNO overlapped, preventing a simultaneous binding of the two inhibitors to the NQR complex. The site of Ag+ inhibition was the initial reaction of the NQR complex catalyzed by Nqr6 subunit. The time courses of Ag+ inhibition and the release of FAD indicate that the Ag+-denatured Nqr6 subunit gradually releases FAD.

Anti-Bacterial Agents↗
PubMed1999

Serum biochemical values in two inbred strains of mastomys (Praomys coucha).

Serum samples collected from 119 (72 male and 47 female) mastomys (Praomys coucha) of 2 specific-pathogen-free inbred strains (RI4 and RI7) were analyzed for 12 serum biochemical parameters. Sex-related differences (p < 0.01) were noted in alkaline phosphatase and glucose; the both higher in females than in males. Age-related changes (p < 0.01) were observed in total protein, albumin, total cholesterol, and alkaline phosphatase, with higher values for the first three parameters in the older group (200-250 days of age) than in the younger group (90-140 days of age). Four out of 12 parameters showed strain-related differences (p < 0.01), consistent with the large amount of genetic heterogeneity reported in this species. These serum biochemical reference values should provide information for the use of mastomys in laboratory research.

Animals↗
PubMed1998

lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal.

lefty-1, lefty-2, and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1-deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1-/- mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal, lefty-2, and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Animals↗
PubMed1998

Isolation of the rat spermatid manchette and its perinuclear ring.

The manchette is a transient structure that develops during spermiogenesis. It consists of three components: a perinuclear ring, a microtubule mantle inserted in the ring, and dense plaques attached at the distal end of the mantle. A procedure has been developed for the fractionation of intact manchettes from rat spermatids. Each fractionation step was monitored by indirect immunofluorescence using an antibody to unmodified alpha-tubulin. Indirect immunofluorescence and electron microscopy demonstrate that fractionated manchettes are relatively intact. A thermocleavage step was used to sever the microtubule mantle from the perinuclear ring. Microtubules of the mantle collected in a stabilizing buffer containing Taxol formed long bundles of side-by-side aligned microtubules. The perinuclear ring sample consisted of circular-shaped units of different diameter with truncated microtubules still attached to the ring, a property that enabled the initial recognition of the rings by alpha-tubulin antibody staining. Indirect immunofluorescence and immunoblotting experiments using isoform-specific antibodies to alpha-tubulins show that the manchette contains acetylated, tyrosinated, glutamylated alpha-tubulin and an alpha-3/7 tubulin isoform. The same alpha-tubulin isoforms were observed in the axoneme of the sperm tail. Two-dimensional polyacrylamide gel electrophoresis fractionation maps of silver-stained proteins of the intact manchette show four predominant proteins: alpha- and beta-tubulins, beta-actin, vimentin, and a 62-kDa protein. The latter persisted in thermocleaved perinuclear ring samples. Results of this study indicate that the newly developed procedure for the fractionation of manchettes will facilitate a direct characterization of posttranslationally modified tubulin variants, microtubule-associatedproteins, and the components of the perinuclear ring of this largely neglected structure of the spermiogenic process.

Animals↗
PubMed1998

Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes.

Primary spermatocytes are the male germ cells before meiosis I. To examine whether these 4n diploid cells are genetically competent to fertilize oocytes and support full embryo development, we introduced the nuclei of pachytene/diplotene spermatocytes into oocytes that were arrested in prophase I (germinal vesicle stage), metaphase I, or metaphase II (Met II). Both the paternal and maternal chromosomes then were allowed to undergo meiosis synchronously until Met II. In the first and second groups, the paternal and maternal chromosomes had intermingled to form a large Met II plate, which was then transferred into a fresh enucleated Met II oocyte. In the third group, the paternal Met II chromosomes were obtained by transferring spermatocyte nuclei into Met II oocytes twice. After activation of the Met II oocytes that were produced, those microfertilized at metaphase I showed the best developmental ability in vitro, and three of these embryos developed into full-term offspring after embryo transfer. Two pups (one male and one female) were proven to be fertile. This finding provides direct evidence that the nuclei of male germ cells acquire the ability to fertilize oocytes before the first meiotic division.

Animals↗
PubMed1998

Regression of cervical disc herniation observed on magnetic resonance images.

STUDY DESIGN: A retrospective study of cervical disc herniation using results of repeated magnetic resonance imaging examinations. OBJECTIVES: To clarify the cervical disc herniation morphological changes over time in order to establish a strategy for treatment. SUMMARY OF BACKGROUND DATA: In the authors' previous magnetic resonance imaging follow-up study of patients with lumbar disc herniation, spontaneous regression was observed in the sequestration-type lesions, and it was found that the tendency toward regression differed based on the anatomic position of extruded disc material. METHODS: Thirty-eight patients with cervical disc herniation who underwent repeated magnetic resonance imaging examinations were studied. The changes over time in herniated disc size were evaluated using this imaging technique. Evaluation showed the characteristics of those in whom spontaneous regression was found, such as extrusion pattern, and the clinical outcome was evaluated by symptoms. RESULTS: In 15 patients (40%), the volume of herniated material was decreased. The interval from onset of symptoms to the initial examination was significantly shorter in the regression group than in the group that showed no change in disc herniation. By extrusion pattern, cervical disc herniation, which was divided into migration type on sagittal view and lateral type on axial view, most frequently exhibited spontaneous regression. All of the patients with radicular pain and upper limb amyotrophy were treated successfully with conservative therapy. CONCLUSION: Although the possibility of the combination of hemorrhage and disc material could not be denied, active resorption of herniated material probably occurred during the acute phase. Extruded material exposed to the epidural space may be resorbed more quickly than that beneath the ligament. Vascular supply probably plays a role in the mechanism of resorption. The phase and position of extrusion were the significant factors affecting cervical disc herniation resorption. It was demonstrated that examination performed during the acute phase using magnetic resonance imaging is necessary for elucidation of the pathogenesis of cervical disc herniation, and that migrating, lateral-type herniations regress so frequently that conservative treatment should be chosen not only for patients with radicular pain, but also for those with upper limb amyotrophy.

Adult↗
PubMed1998

Actions of cyclic esters, S-esters, and amides of phenyl- and phenylthiophosphonic acids on mammalian and insect GABA-gated chloride channels.

Cyclic esters, S-esters, and amides of phenyl(thio)phosphonic acid were synthesized to probe the interaction between noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors and their binding site. Some of these compounds competitively inhibited the specific binding of [3H]EBOB, a noncompetitive GABA antagonist, to rat-brain and housefly-head membranes. The trans isomer of the ester bearing a tert-butyl group at the 5-position and a bromine atom at the p-position (5t) was most potent in rat receptors with an IC50 value of 40 nM, while the trans isomer of the S-ester bearing the same substituents (10t) was most potent in housefly receptors with an IC50 value of 55 nM. In both cases, the corresponding amide analogue (12t) was less potent. The potencies of 5t and 12t tended to decrease in the presence of GABA, particularly in housefly receptors, while that of 10t remained unchanged. The rank order of activity in inhibiting [3H]EBOB binding to housefly-head membranes in the presence of GABA (10t > 5t > 12t) was in accord with that of insecticidal activity. S-Ester 10t depressed 10 microM and 300 microM GABA-induced 36Cl- influx into mouse cerebral synaptoneurosomes, whereas ester 5t depressed 10 microM GABA-induced 36Cl- influx but not 300 microM GABA-induced flux. Amide 12t was inactive at both GABA concentrations. These findings indicate that six-membered cyclic phenylthiophosphonic acid derivatives act as noncompetitive antagonists of GABA receptors and suggest that 10t is able to bind to the receptor in the open, desensitized, and closed states, whereas the affinity of 5t and 12t is lower in the open and desensitized states than in the closed state. The derivatives have similar structures except for the heteroatoms at the 1- and 3-positions, so that the heteroatoms may play a unique role when antagonists bring the open state of the GABA-gated channel to the desensitized or closed state.

Affinity Labels↗
PubMed1998

Picrodendrin and related terpenoid antagonists reveal structural differences between ionotropic GABA receptors of mammals and insects.

Twenty-eight picrotoxane terpenoids, including picrodendrins isolated from the Euphorbiaceae plant, Picrodendron baccatum (L.) Krug and Urban, have been evaluated for their ability to inhibit the specific binding of [3H]EBOB, the noncompetitive antagonist of ionotropic GABA receptors, to rat-brain and housefly (Musca domestica L.)-head membranes. Picrodendrin Q was the most potent competitive inhibitor of [3H]EBOB binding, with IC50 values of 16 nM (rat) and 22 nM (Musca). We find that the spiro gamma-butyrolactone moiety at the 13-position, which contains a carbonyl group conjugated with an unsaturated bond, and the substituents at the 4-position play important roles in the interaction of picrodendrins with their binding site in rat receptors. In contrast, such structural features are not strictly required in the case of the interaction with Musca receptors; the spiro saturated gamma-butyrolactone moiety at the 13-position, which bears the 16-sp3 carbon atom, and the hydroxyl groups at various positions are somewhat tolerated. Quantitative structure-activity studies have clearly shown that the electronegativity of the 16-carbon atom and the presence or absence of the 4- and 8-hydroxyl groups are important determinants of the potency of nor-diterpenes in Musca receptors, while the negative charge on the 17-carbonyl oxygen atom is likely important in the case of rat receptors. These findings indicate that there are significant differences between the structures of the complementary binding sites in rat GABA receptors and Musca GABA receptors. We also infer differences between native Musca GABA receptors and the Drosophila Rdl subunit-containing homo-oligomeric GABA receptors in the structures of their binding sites.

Animals↗