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Biomedical subjects

M Ito

Publications and source records attributed to M Ito.

At least 307 records · Page 17Linked to original sources
PubMed2001

Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle.

Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.

Animals↗
PubMed2001

Induction of long-term depression in cerebellar Purkinje cells requires a rapidly turned over protein.

Evidence is presented indicating that the induction of long-term depression (LTD) in Purkinje cells (PCs) requires a rapidly turned over protein(s) during a critical time period within 15 min after the onset of LTD-inducing stimulation and that synthesis of this protein is maintained by mRNAs supplied via transcription. LTD was induced in granule cell axon (GA)-to-PC synapses by stimulation of these synapses at 1 Hz for 5 min in conjunction with the climbing fibers (CFs) forming synapses on the same PCs and represented by a persistent reduction in the GA-induced excitatory postsynaptic potentials (EPSPs). Not only a prolonged but also a brief (5 min) pulse application of translational inhibitors (anisomycin, puromycin, or cycloheximide) effectively blocked the LTD induction. Pulses applied during the period from 30 min before to 10 min after the onset of conjunctive stimulation blocked the LTD induction, but those applied 15 min after were ineffective. The three translational inhibitors blocked the LTD induction similarly, suggesting that the effect is due to their common action of inhibiting protein synthesis. Infusion of a mRNA cap analogue (7-methyl GTP) into PCs also blocked LTD induction, ensuring that the postsynaptic protein synthesis within PCs is required for LTD induction. Transcriptional inhibitors, actinomycin D and 5,6-dichloro-l-beta-D-ribofuranosyl-benzimidazole, also blocked the LTD induction, but this effect was apparent when 5-min pulses of the transcriptional inhibitors preceded the conjunctive stimulation by 30 min or more. This time lag of 30 min is presumed to be required for depletion of the protein(s) required for LTD induction. The presently observed effects of translational and transcriptional inhibitors on the LTD induction are of temporal characteristics corresponding to their depressant effects on the type-1 metabotropic glutamate-receptor (mGluR1)-mediated slow EPSPs in PCs as we have reported recently. An antagonist of mGluR1s [(RS)-1-aminoindan-1,5-dicarboxylic acid], however, did not block LTD induction when it was applied during the 10-min period following conjunctive stimulation, where translational inhibitors effectively blocked LTD induction. This discrepancy in time course suggests that the rapidly turned over protein(s) required for LTD induction is involved in a process occurring downstream of the activation of mGluR1s.

Animals↗
PubMed2001

Cerebellar long-term depression: characterization, signal transduction, and functional roles.

Cerebellar Purkinje cells exhibit a unique type of synaptic plasticity, namely, long-term depression (LTD). When two inputs to a Purkinje cell, one from a climbing fiber and the other from a set of granule cell axons, are repeatedly associated, the input efficacy of the granule cell axons in exciting the Purkinje cell is persistently depressed. Section I of this review briefly describes the history of research around LTD, and section II specifies physiological characteristics of LTD. Sections III and IV then review the massive data accumulated during the past two decades, which have revealed complex networks of signal transduction underlying LTD. Section III deals with a variety of first messengers, receptors, ion channels, transporters, G proteins, and phospholipases. Section IV covers second messengers, protein kinases, phosphatases and other elements, eventually leading to inactivation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate-selective glutamate receptors that mediate granule cell-to-Purkinje cell transmission. Section V defines roles of LTD in the light of the microcomplex concept of the cerebellum as functionally eliminating those synaptic connections associated with errors during repeated exercises, while preserving other connections leading to the successful execution of movements. Section VI examines the validity of this microcomplex concept based on the data collected from recent numerous studies of various forms of motor learning in ocular reflexes, eye-blink conditioning, posture, locomotion, and hand/arm movements. Section VII emphasizes the importance of integrating studies on LTD and learning and raises future possibilities of extending cerebellar research to reveal memory mechanisms of implicit learning in general.

Animals↗
PubMed2001

Point mutation (-69 G-->A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B-containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the -69 nucleotide of the promoter region (-69 G-->A), corresponding to the second nucleotide of the PEA3/ETS binding site (CGGAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol >/=2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the -69 G-->A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the -69 G-->A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel -69 G-->A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.

Carrier Proteins↗
PubMed2001

Microsatellite instability in transforming growth factor-beta 1 type II receptor gene in alveolar lining epithelial cells of idiopathic pulmonary fibrosis.

It has been reported that transforming growth factor (TGF)-beta, which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the growth-inhibitory effects of TGF-beta, we searched for mutations in the microsatellite of the TGF-beta receptor type II (T beta RII) gene. To detect a deletion in the polyadenine tract in exon 3 of the T beta RII gene, cells were isolated by microdissection from lung sections of IPF patients, and DNA was extracted from these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low T beta RII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite instability in the T beta RII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this genetic disorder might play a partial role in the pathologic changes of IPF.

Adult↗
PubMed2001

Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.

DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.

Animals↗
PubMed2001

Sertoli cell-specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice.

DAX1 is an orphan member of the nuclear hormone receptor superfamily of transcription factors. Our recent characterization of Dax1 (Ahch)-deficient male mice revealed a primary testicular defect resulting in hypogonadism and sterility. The progressive degeneration of the germinal epithelium, independent of abnormal gonadotropin and testosterone production, suggested an intrinsic loss of Dax1 function in the Sertoli cells. To test this hypothesis, we assessed the effect of Sertoli cell-specific expression of a human DAX1 (AHC) transgene driven using the promoter of the Müllerian inhibiting substance (MIS) gene. The MIS-DAX1 transgene partially rescued the mutant phenotype of the Dax1-deficient male mice. Although testicular morphology remained abnormal, fertility was restored to levels matching that of wild-type littermates. Examination of several markers of sperm fertilizing capability revealed significant improvements in MIS-DAX1-rescued mice. Epididymal sperm count and sperm motility were greater in 12-week-old rescued mice than in age-matched Dax1-deficient mice. The ability of sperm to undergo an immediate acrosome reaction was impaired in Dax1-deficient animals, and sperm from Dax1-deficient mice fertilized only 8.2 +/- 6.8% of eggs in vitro, significantly less than rescue (67.8 +/- 19.1%) and wild-type (88.9 +/- 3.9%) sperm. These results indicate that Dax1 expression in Sertoli cells is adequate to overcome crucial thresholds related to sperm production and function. However, the failure to completely rescue the testicular pathology of Dax1-deficient mice suggests that Dax1 expression in other somatic cells is essential for normal testicular development.

Acrosome Reaction↗
PubMed2001

Missense mutations cluster within the carboxyl-terminal region of DAX-1 and impair transcriptional repression.

DAX-1 is an orphan nuclear receptor that plays a key role in the development and function of the adrenal gland and hypothalamic-pituitary gonadal axis. Mutations in the gene encoding DAX-1 result in X-linked adrenal hypoplasia congenita (AHC). Affected boys typically present with primary adrenal failure in infancy or childhood and hypogonadotropic hypogonadism at the time of puberty. The majority of DAX1 mutations described to date are nonsense or frameshift mutations that result in premature truncation of the DAX-1 protein and loss of DAX-1 repressor function. Relatively few missense mutations in DAX1 have been reported. Here, we describe missense mutations in three additional families with X-linked AHC. When combined with previous reports, the DAX1 missense mutations appear to cluster within restricted regions of the putative ligand-binding domain of DAX-1 and affect amino acids that are evolutionarily conserved, suggesting that these regions correspond to critical functional domains. Transcription assays, using a variety of artificial and native target genes, were performed to assess the effects of these mutations on the function of DAX-1. All DAX-1 missense mutant constructs showed marked loss of repressor function, with the exception of I439S, a mutation previously shown to be associated with delayed-onset adrenal failure and incomplete hypogonadotropic hypogonadism. These data indicate that most DAX1 missense mutations associated with classic AHC exhibit marked loss of function. The locations of these mutations thereby identify important functional domains in the carboxyl-terminus of the protein.

17-alpha-Hydroxyprogesterone↗
PubMed2001

N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia.

WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42.

Actins↗
PubMed2001

Molecular cloning, functional expression and characterization of (E)-beta farnesene synthase from Citrus junos.

We cloned the gene of the acyclic sesquiterpene synthase, (E)-beta-farnesene synthase (CJFS) from Yuzu (Citrus junos, Rutaceae). The function of CJFS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. CJFS consisted of 1867 nucleotides including 1680 bp of coding sequence encoding a protein of 560 amino acids with a molecular weight of 62 kDa. The deduced amino acid sequence possessed characteristic amino acid residues, such as the DDxxD motif, which are highly conserved among terpene synthases. This is the first report of the cloning of a terpene synthase from a Rutaceous plant. A possible reaction mechanism for terpene biosynthesis is also discussed on the basis of sequence comparison of CJFS with known sesquiterpene synthase genes.

Amino Acid Sequence↗
PubMed2001

Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia.

Limonene is one of the most simple cyclic monoterpenes, and two enantiomers, d- and l-limonene occur due to the chiral carbon at 4-position. Cyclization of GPP into limonene is catalyzed by the limonene synthase, and some l-limonene synthase cDNAs have already been cloned from several species of plants, mainly from Labiatae family. However, the d-limonene synthase gene has not yet been obtained, therefore, no information is available on the molecular mechanism of stereochemical regulation in limonene formation. To resolve this, we cloned the d-limonene synthase gene (dLMS) from Schizonepeta tenuifolia (Labiatae) by a reverse genetic approach, and we found that both d- and l-limonene synthase share similar features such as a transit peptide, an arginine rich domain, and a metal cation binding site in their structures. Here, we report on the cloning of dLMS, and the putative stereochemical regulation mechanism is discussed based on the comparison of the deduced amino acid sequence of dLMS with those of known l-limonene synthases.

Amino Acid Sequence↗
PubMed2001

Evaluation of insulin permeability and effects of absorption enhancers on its permeability by an in vitro pulmonary epithelial system using Xenopus pulmonary membrane.

The permeability of insulin across Xenopus pulmonary membrane and the effects of various absorption enhancers on insulin permeability were examined using an in vitro Ussing chamber technique. Absorption enhancers used in this study were sodium caprate (NaCap), sodium glycocholate (NaGC), sodium salicylate (NaSal) and ethylenediaminetetraacetic acid disodium salt (EDTA). The permeability of insulin across Xenopus pulmonary membrane significantly increased in the presence of NaCap and NaGC, while EDTA and NaSal did not enhance the permeability. In addition, the enhancing effect of NaGC increased as the concentrations of these enhancers increased. Transmembrane resistance (Rm) of Xenopus lung was markedly decreased in the presence of these enhancers, and NaCap showed a greater effect on Rm than NaGC. Furthermore, the amount of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) released from the apical side of the Xenopus pulmonary membrane increased in the presence of these enhancers. These results indicate that NaCap and NaGC improve the pulmonary absorption of insulin, but they are toxic to the pulmonary membrane. These findings suggest that this method is useful for estimating the permeability characteristics of peptides across the pulmonary membrane and for evaluating the effects of various additives on their permeability and their membrane toxicity.

Absorption↗
PubMed2001

Phlomisflavosides A and B, new flavonol bisglycosides from Phlomis spinidens.

From the aerial parts of Phlomis spinidens, two new flavonol bisglycosides, phlomisflavosides A (1) and B (2), were isolated together with the known compounds, astragalin, isoquercitrin, lamiridoside, phlomoside A, shanzhiside methyl ester, 8-O-acetylshanzhiside methyl ester, phlorigidoside C, rodioloside (=salidroside), forsythoside B, citroside A and lariciresinol-4'-O-beta-D-glucoside. The structures of the new compounds were elucidated based on spectral and chemical evidence.

Flavonoids↗
PubMed2001

Farnesyl hydroxybenzoic acid derivatives from Ferula kuhistanica.

Four new farnesyl hydroxybenzoic acid derivatives, kuhistanols E-H (1-4), were isolated from the roots of the Uzbekistan medicinal plant Ferula kuhistanica. The structures of the new compounds were elucidated based on spectroscopic and chemical evidence.

Apiaceae↗
PubMed2001

Chemical constituents of Prangos tschiniganica; structure elucidation and absolute configuration of coumarin and furanocoumarin derivatives with anti-HIV activity.

The methanol extract of the dried aerial parts of Prangos tschimganica gave three new coumarin derivatives and 30 known coumarin derivatives. Their structures were established on the basis of chemical and spectroscopic evidence. Absolute configuration of the isolated compounds were determined by using a modified Mosher's method. Some of the isolated compounds showed anti-HIV activity.

Acetylation↗
PubMed2001

Level of plasma tissue factor pathway inhibitor is inversely correlated with intraarterial diastolic pressure in subjects who underwent coronary angiography.

There are only a few studies of the relationship between hemostatic abnormalities and intraarterial pressure, so the present study investigated the association of various newer lipid and hemostatic variables with intraarterial pressure levels. Levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, triglyceride, lipoprotein-(a), remnant-like particle cholesterol, cholesteryl ester transfer protein, uric acid, blood glucose, fibrinogen, free form of tissue factor pathway inhibitor (TFPI), C-reactive protein, serum amyloid A protein, anti-Chlamydia pneumoniae immunoglobulin G and immunoglobulin A, and apolipoproteins (apo) A-I, B, and E were measured in 176 patients who underwent diagnostic coronary angiography. Intraarterial blood pressure was determined from central aortic pressure using a standard fluid-filled catheter-external transducer system. Multivariate regression analyses showed that TFPI level was the only independent factor associated with aortic diastolic pressure. The linear regression equation demonstrated a significant negative correlation of TFPI level with aortic diastolic pressure (r=-0.395, p=0.0011). With respect to the association with other parameters, the TFPI level showed significant correlations between the HDL-C level and the apo A-I level, both in the overall patients and in the patients with coronary artery stenosis. This is the first evidence that the level of the plasma free form of TFPI is inversely correlated to aortic diastolic pressure.

Adult↗