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Biomedical subjects

N Ishida

Publications and source records attributed to N Ishida.

At least 127 records · Page 7Linked to original sources
PubMed1998

The role of transcription factors in circadian gene expression.

There are three basic components present in all species which are essential for the circadian gene expression; an input pathway which connects the clock to the environment, the clock oscillator, and an output pathway which connects the pacemaker to the resulting biological phenomena. In this review, an attempt to separate the three processes will be made from the molecular biological stand point. In the pineal of birds, Drosophila, and algae, cAMP/PKA pathway is functional in the output, but in mammalian suprachiasmatic nucleus (SCN), this pathway including the CREB/c-fos pathway, is believed to function in the input pathway. We propose here a model that easily explains the controversial results of the CREB/c-fos story in the SCN by considering this signal transduction pathway as an output. Finally, we propose the importance of E-box and bHLH-PAS type transcription factors, in the clock oscillator in the SCN.

Animals↗
PubMed1998

Examination of DNA-binding activity of neuronal transcription factors by electrophoretical mobility shift assay.

Electrophoretical mobility shift assay (EMSA) is a simple, rapid, and highly sensitive technique for detection of single- or double-stranded DNA-binding proteins such as transcription factors in crude nuclear extracts (F.M. Ausubel, R. Brent, R.E. Kingston, D. D. Moore, J.G. Seidman, J.A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, 1989, pp. 12.0.1-12.2.10 [1]; J. Carey, Gel Retardation. Methods Enzymol., 208 (1991) 103-117 [2]). By using this technique, it is possible to quantify the abundance, relative affinity and binding specificity of DNA-binding proteins. Since proteins which bind specifically to radiolabeled DNA probes retard the mobility of the probe during electrophoresis (it also called gel retardation assay), discrete bands correspond to the individual DNA-protein complexes. Furthermore, EMSA allows one to determine which member(s) of a certain protein family are included in the DNA-protein complex by means of specific antibodies raised against the DNA-binding protein (supershift assay).

Animals↗
PubMed1998

Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies.

Nucleotide sugar transporters are mainly located in the Golgi membranes and carry nucleotide sugars, that are produced outside the Golgi apparatus, into the organelle, where they serve as substrates for the elongation of carbohydrate chains by glycosyltransferases. They are thus indispensable for cellular glycoconjugate synthesis and, moreover, may have regulatory roles in producing the structural variety of cellular glycoconjugates. Their occurrence has long been well recognized, but studies on the molecular bases of their strict substrate specificities and modes of action have been hampered by the lack of information on their precise molecular structures. Complementary DNAs encoding several of these transporters were cloned recently, which represented a substantial step forward as to the above mentioned issues. The products of these cDNAs are mutually related hydrophobic proteins consisting of 320-400 amino acid residues with multiple putative transmembrane helix domains, and are located in the Golgi apparatus. This review briefly summarizes the present status of the field of nucleotide sugar transporter research, and also presents an outlook of the study in this field.

Amino Acid Sequence↗
PubMed1998

Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae.

We describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae. Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter. The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus. The hUGT product was expressed in the membranes of Golgi and other organellar compartments. The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity. The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 microM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles. Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles. Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins. The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters.

Biological Transport↗
PubMed1998

Functional expression of human golgi CMP-sialic acid transporter in the Golgi complex of a transporter-deficient Chinese hamster ovary cell mutant.

We recently described the cloning of putative human CMP-sialic acid transporter (hCST) cDNA [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. The hCST cDNA coded for a hydrophobic protein with an amino acid sequence showing a high degree of similarity (92% identity) to that of murine CMP-sialic acid transporter. In this report, we demonstrate that hCST corrects the CMP-sialic acid transporter-deficient phenotype of CHO-derived Lec2 cells, as judged from the recovery of WGA-sensitivity by transformants, and the recovery of CMP-sialic acid transporting ability by microsomal vesicles prepared from them. A peptide antibody against the C-terminus of the hCST protein detected the cDNA products expressed in the microsomes of the transformants. The subcellular localization of the hCST protein in the Golgi membrane was demonstrated by immunofluorescence microscopy, using the hCST-specific antibody. These results clearly indicate that hCST cDNA encodes the human CMP-sialic acid transporter protein. Plasma membrane-selective permeabilization combined with immunofluorescence microscopy provided strong evidence that the C-terminus of the human CMP-Sia transporter is exposed to the cytosol on the outer surface of the Golgi membrane.

Animals↗
PubMed1998

Isolation and characterization of a novel connexin gene, Cx-60, in porcine ovarian follicles.

A new member of the connexin family was isolated from the porcine ovary. The amino acid sequence deduced from the nucleotide sequence of genomic as well as complementary DNA clones predicted the reading frame encoding the 60-kDa protein product, indicating the largest molecular mass among the connexin family genes analyzed to date; we named this gene Cx-60 based on its predicted molecular mass. The features of its primary structure were compared with those of other connexin genes, and the characteristics of its expression profile were examined in mammalian ovarian follicles. Cx-60 shares significant similarities with other connexins in the transmembrane and extracellular domains, but showed a highly unique primary structure in the cytoplasmic and carboxyl-terminal domains. The Cx-60 gene was unique in that its messenger RNA was detected in both the theca interna compartment and cumulus cells in the ovary; of other tissues, Cx-60 expression was relatively evident in the colon, thymus, and spleen. Cx-60, in contrast to Cx-43, was expressed constitutively upon gonadotropin stimulation when examined in hypophysectomized rats. Taken together, these results indicate that at least the connexin genes Cx-60, Cx-43, Cx-32, Cx-30.3, and Cx-26 are expressed in porcine ovarian follicles with differing expression profiles, including cell specificity.

Amino Acid Sequence↗
PubMed1998

[A case of Marshall syndrome with secondary glaucoma due to spontaneous rupture of the lens capsule].

An eight-year-old boy developed acute glaucoma in his left eye. He was diagnosed as having Marshall syndrome because of bilateral cataract, high myopia, saddle nose, hypertelorism, and lack of arthropathy. When seen immediately after the glaucomatous attack, the affected eye showed ruptured anterior and posterior lens capsules and displacement of the lens nucleus into the anterior chamber and vitreous cavity. Lens extraction and vitrectomy induced normalization of intraocular pressure and visual acuity. There are forty cases reported in the world literature and only one cases in Japan. To the best of our knowledge, this is the first case treated successfully by vitreous surgery.

Acute Disease↗
PubMed1998

[Running fit and generalized tonic-clonic seizure are differently controlled by different subtype receptors in the brainstem].

Rats neonatally treated with 0.02% propylthiouracil (PTU) through mother's milk showed a high incidence of audiogenic seizures after maturation. These audiogenic seizures were differently modified by MK-801 and NBQX; while intraperitoneal MK-801 equally inhibited running fit (RF) and generalized tonic-clonic seizure (GTCS), NBQX administered into cisterna ambiens significantly inhibited RF but not GTCS. The possible involvement of glutamate receptors in the inferior colliculus was further investigated using naive Sprague-Dawley rats injected with NMDA, AMPA or cyclothiazide, known as an inhibitor of desensitization of AMPA action. All drugs tested successfully induced RF followed by GTCS, resembling audiogenic seizures in PTU-treated rats. However, sound stimulation could augment AMPA-induced, but not NMDA-induced GTCS. Systemic administration with MK-801 potently blocked GTCS induced by AMPA/cyclothiazide, but the same drug failed to block RF after intracisternal injection with AMPA/cyclothiazide. Furthermore, intracisternal administration with NBQX significantly inhibited only RF induced by AMPA/cyclothiazide. The present study suggests that: 1) glutamate receptors in the brainstem, possible in the inferior colliculus, play a crucial role in audiogenic seizures, namely the initiation of RF and propagation into GTCS; and 2) the initiation mechanism is regulated by both NMDA and AMPA receptors, whereas propagation is mainly controlled by NMDA receptors.

Acoustic Stimulation↗
PubMed1997

Molecular cloning and expression of a novel human aquaporin from adipose tissue with glycerol permeability.

In a systematic analysis of genes expressed in human adipose tissue, we detected a novel gene that is expressed uniquely in adipose tissue. The sequence showed that it encodes a 342-amino-acid protein containing six putative transmembrane domains, and is a new member of the aquaporin family of water-selective membrane channels. We named this gene aquaporin 9. It features a cyclic-AMP protein kinase phosphorylation consensus site in the NH3-terminal domain. Expression of the cRNA in Xenopus oocytes yielded a 7-fold increase in osmotic water permeability blocked by 0.3 mM HgCl2, and also facilitated the uptake of glycerol. Northern blot analysis demonstrated that the mRNA is abundant in adipose tissue, but not in other tissues. Thus, this gene product may participate in glycerol transport in adipocytes.

Adipose Tissue↗
PubMed1997

Immunolocalization of aquaporin homologs in mouse lacrimal glands.

It was shown recently that the aquaporin family of water channels exists in lacrimal gland cells. To determine the localization of aquaporin homologs and the pathophysiological modification of aquaporins by pilocarpine, a muscarinic agonist, we performed immunohistochemistry and immunoblotting in mouse lacrimal glands. By immunohistochemistry, aquaporin-4 (AQP4) and aquaporin-5 (AQP5) were found to be the lacrimal glands. AQP5 immunolabeling was detected in the apical membranes of the acinus and duct cells, while AQP4 was expressed in the basolateral membranes only. The tear secretion of mice systemically treated with pilocarpine was significantly (2.5-fold) higher than that of the saline-treated controls. The antibody to the AQP5 carboxy terminus showed high immunoreactivity on the apical membrane in the pilocarpine-treated lacrimal glands but not on that of saline-treated controls. However, the antibody to the extracellular domain of AQP5 showed similar immunolabeling in both groups of animals. In contrast, the immunoreactivity of AQP4 was not affected by pilocarpine stimulation. As shown by western blot analysis, the expression level of AQP5 on the apical membrane in the pilocarpine-stimulated lacrimal glands was not significantly different compared with the saline-treated controls. We conclude that AQP4 and AQP5 water channels are expressed on mouse lacrimal gland cells, with greater expression of AQP4 on the basolateral membrane and of AQP5 on the apical membrane. Furthermore, the AQP5 carboxy terminus region may undergo pathophysiological modification when tear secretion is increased by pilocarpine stimulation.

Animals↗
PubMed1997

KMD-3213, a novel alpha1A-adrenoceptor antagonist, potently inhibits the functional alpha1-adrenoceptor in human prostate.

KMD-3213, (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluoroethoxy)phe noxy]ethyl]amino]propyl]indoline-7-carboxamide, is a novel and selective alpha1A-adrenoceptor antagonist. The potency of this drug to antagonize functional alpha1-adrenoceptor-mediated contraction in human prostatic smooth muscle was evaluated and compared with that of other alpha1-adrenoceptor antagonists. KMD-3213 inhibited noradrenaline-induced contractions with an apparent pK(B) value of 9.45 +/- 0.039, indicating a potency similar to that of tamsulosin. The affinity of prazosin for prostatic alpha1-adrenoceptors is given as potency for the alpha1L-adrenoceptor with an estimated pA2 value of 8.84 +/- 0.044. The data obtained in this study suggest that KMD-3213, an alpha1A-adrenoceptor-selective antagonist, has strong affinity for the alpha1L-adrenoceptor in the human prostate.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1997

Impaired fertility in mice deficient for the testicular germ-cell protease PC4.

PC4 is a member of the proprotein convertase family of serine proteases implicated in the processing of a variety of polypeptides including prohormones, proneuropeptides, and cell surface proteins. In rodents, PC4 transcripts have been detected in spermatocytes and round spermatids exclusively, suggesting a reproductive function for this enzyme. In an effort to elucidate this function, we have disrupted its locus (Pcsk4) by homologous recombination in embryonic stem cells and have produced mice carrying the mutation. In intercrosses of heterozygous mutant mice, there was low transmission of the mutant Pcsk4 allele to the progeny, resulting in lower than expected incidence of heterozygosity and null homozygosity. The in vivo fertility of homozygous mutant males was severely impaired in the absence of any evident spermatogenic abnormality. In vitro, the fertilizing ability of Pcsk4 null spermatozoa was also found to be significantly reduced. Moreover, eggs fertilized by these spermatozoa failed to grow to the blastocyst stage. These results suggest that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.

Animals↗
PubMed1997

Purification, cDNA cloning, and characterization of a new serpin with megakaryocyte maturation activity.

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

Amino Acid Sequence↗
PubMed1997

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.

Amino Acids↗
PubMed1997

Efficient gene expression in mammalian clock pacemaker cell in vitro by an adenovirus vector.

An efficient modified adeno system to express foreign genes to the central nervous system was developed recently. This modified recombinant adenoviral vectors can be used successfully to deliver lacZ to the hypothalamic suprachiasmatic nucleus (SCN) which is composed of mammalian clock pacemaker. The expression of lacZ in the primary culture of SCN was dose-dependent and higher enough in nearly 100% of these cells. We also showed that viral toxicity and lacZ overexpression had no serious effects on the rhythmic expression of arginine vasopressin (AVP) release from the SCN cell culture.

Adenoviridae↗
PubMed1997

Regulation of cAMP response element binding protein (CREB) binding in the mammalian clock pacemaker by light but not a circadian clock.

Mammalian circadian rhythms are considered to be regulated by a clock pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanism of entrainment and oscillation of circadian rhythm are not well understood but photic induction of immediate-early gene (IEG) expression in the SCN is thought to play a role. Here we show that under 12 h light:12 h dark (LD) condition, the cAMP response element binding protein (CREB) binding to cAMP responsive promoter element (CRE) of NMDAR1/zeta1 promoter region in the SCN is higher during the light than the dark by electro-mobility shift assay (EMSA). When animals are placed in constant dark, CREB DNA binding activity in the SCN is low and does not vary with circadian time when compared with cortex nuclear extract as a control. Most significantly, photic induction of CREB binding activity in the SCN occurs at all circadian times tested, indicating that CREB DNA binding in the SCN is not gated by the endogenous clock. These results implicate the role of CREB in photic neuronal signaling in the SCN and suggest that CREB DNA binding activities may not be regulated by a circadian clock.

Animals↗
PubMed1997

Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment.

The effects of trimethyltin on the hippocampus were investigated in terms of changes in histology, depth electroencephalography, learning acquisition and memory retention, choline acetyltransferase and neuropeptides, and seizure-induced c-fos messenger RNA expression. The results were as follows. (1) Morphologically, trimethyltin produced a progressive loss of hippocampal CA3 and CA4 pyramidal cells, starting from four days after peroral treatment with trimethyltin hydroxide (9 mg/kg), as described previously. (2) Neurophysiologically, the increased seizure susceptibility to pentylenetetrazol treatment reached a maximum at four days post-trimethyltin and then declined after five days post-trimethyltin. The maximal seizure susceptibility at four days post-trimethyltin was confirmed by the immediate and long-lasting appearance of spike discharge in the hippocampus. However, this was not verified by the expression of c-fos messenger RNA in the hippocampus, which was comparable between trimethyltin-treated and control rats. (3) Behaviorally, the time-courses of aggression and learning impairment were similar to that of the seizure susceptibility. (4) Neurochemically, trimethyltin treatment caused changes of neurochemical markers, which were manifested by the elevation of neuropeptide Y content in the entorhinal cortex, and of choline acetyltransferase in the hippocampal CA3 subfield. Trimethyltin may offer potential as a tool for investigations on the relationship between neuronal death in the hippocampus and the development of seizure susceptibility and learning impairment. Alterations in glucocorticoids, glutamate and neuropeptides may all contribute to the manifestation of the trimethyltin syndrome.

Animals↗