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T Nishigaki

Publications and source records attributed to T Nishigaki.

At least 55 records · Page 3Linked to original sources
PubMed1996

Guanine-rich oligonucleotide modified at the 5' terminal by dimethoxytrityl residue inhibits HIV-1 replication by specific interaction with the envelope glycoprotein.

Previous studies have shown that a guanine-rich oligonucleotide SA-1042, DmTr-TGGGAGGTGGGTCTG, neutralizes HIV-1 infectivity, blocks syncytium formation and inhibits the binding of recombinant gp120 to immobilized soluble CD4 in vitro (Furukawa et al., 1994). We have now investigated the precise mode of action of SA-1042. We show here that SA-1042 specifically antagonizes the binding of anti-V3 loop antibodies or anti-CD4 binding-site antibodies to recombinant gp120, and also blocks the binding of an anti-V3 loop antibody to the V3 peptide (gp120IIIB: aa302-324). In contrast, SA-1042 does not inhibit gp120 binding of monoclonal antibodies directed to other regions of gp120, such as the conserved N-terminal regions (gp120IIIB: aa35-108 or gp120IIIB: aa72-130) or the C-terminal region (gp120IIIB: aa481-496). Furthermore, SA-1042 does not interfere with the binding of monoclonal antibodies directed to other molecules, gp41, CD4, CD11a, CD18, CD26, CD44 or CD54. These data suggest that SA-1042 exerts its antiviral effects by targeting the V3 loop as well as the CD4 binding site on gp120.

Amino Acid Sequence↗
PubMed1996

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA--II. Modification of pyrrolidine ring at P1' proline.

Systematic replacement in the 3- or 4-position of the pyrrolidine ring at P1' proline was carried out. Compound 26, which has a Cl atom in the 4(S)-position was the most active among inhibitors substituted with other halogen atoms or other substituents. Furthermore, the replacement of the Z group in compound 26 with five- or six-membered fused aromatic heterocycle carbonyl groups produced more potent inhibitors. 7-Methoxybenzofuran-2-carbonyl derivative (44) was the best of these and showed Ki = 4.5 nM against HIV PR and IC90S 0.58 microM and 0.06 microM in chronic and acute infections, respectively. These results suggest that the combination of the 4(S)-CI atom and fused bicyclic heterocycles may be effective in improving their cellular penetration.

Cells, Cultured↗
PubMed1996

Structure and function of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs.

Jelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish, Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8 and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.

Acrosome↗
PubMed1996

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease.

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG-->TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3'-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Amino Acid Sequence↗
PubMed1995

Molecular defects in Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive neurodegenerative disorder that affects both the central and peripheral nervous systems due to an enzymatic defect of the galactocerebrosidase. In this study, molecular defects in Krabbe disease were investigated in 11 patients (seven Japanese and four non-Japanese) using cultured skin fibroblasts. A Japanese late infantile patient had a missense mutation of Pro at codon 302 to Ala and a non-Japanese patient had a missense mutation of Val at codon 550 to Gly. The reduced enzymatic activities expressed from the cDNAs with these missense mutations and from the previously reported nonsense mutation (E369X, Glu at codon 369 to stop codon) were confirmed. Genomic DNA analyses revealed that the P302A and E369X mutations were heterozygous and the V550G mutation was homozygous in these patients. A 12 base deletion with a 3 base insertion was found in three unrelated Japanese infantile patients, but not in 30 controls. The mutation was homozygous in two patients and heterozygous in one patient. We could not find any confirmed mutation in the coding region in the other six patients. These findings suggest that mutations in infantile and late infantile patients are relatively heterogeneous.

Amino Acid Sequence↗
PubMed1995

Design and anti-HIV-1 activity of ribozymes that cleave HIV-1 LTR.

A hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the long terminal repeat (LTR) of HIV-1. When the ribozyme catalyzed RNA cleavage reaction for a chemically synthesized 19 mer (LTR 19) was measured, the t 1/2 value of LTR 19 mediated by RZ115 was smaller than that of the RZ119 case. Moreover, the transformed CEM cells harboring the gene encoding these ribozymes were challenged with a HIV-1IIIB strain, two ribozymes, HR112 and RZ119 possessed strong anti-HIV-1 activity. However, the anti-HIV-1 activity displayed by RZ115 was weak. On the basis of secondary structure predictions of the RNA transcribed with the gene encoding ribozymes, the secondary structure of the transcribed RNA with RZ115 sequences was observed to be different from those with the other ribozymes. It has been demonstrated that the secondary structures of transcribed RNAs can possibly influence the anti-HIV-1 activity.

Antiviral Agents↗
PubMed1994

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA.

A series of Human Immunodeficiency Virus type-1 protease (HIV-1 PR) inhibitors that contain 3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA) at the scission site of the substrate were prepared and evaluated for their inhibitory activity. Preliminary studies on the chain length of inhibitors and the hydroxyl configuration of AHPBA indicated that small (2S,3S)-derivatives, composed of the regions between the P3 and P2' sites, showed enough inhibitory activity toward HIV-1 PR to become prototypes for further structural modification. Systematic replacement at the sites from P3 to P2' revealed that some bicyclic heteroarylcarbonyl derivatives possessed strong potency and good enzyme selectivity.

Amino Acid Sequence↗
PubMed1994

Studies of human immunodeficiency virus type 1 (HIV-1) protease inhibitors. III. Structure-activity relationship of HIV-1 protease inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere.

Systematic replacement of the P4-P2 subsites of substrate-based human immunodeficiency virus type 1 protease (HIV-1 PR) inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere (Cha-psi [H.E.]-Ala) at positions corresponding to the scissile sites of substrates was carried out. The structure-activity relationship revealed that compounds with the combination of hydrophilic P3 and beta-branched hydrophobic P2 amino acids generally showed strong inhibitory activity against HIV-1 PR. In particular, compounds 4 (Boc-Orn-Val-Cha-psi [H.E.]-Ala-NHBun; Bu(n) = n-butyl, Ki = 11 nM) and 6 (Z-Orn-Val-Cha-psi [H.E.]-Ala-NHBun, Ki = 8 nM) exhibited good enzyme selectivity, possessing no significant inhibitory activities toward closely related aspartic proteases, pepsin, cathepsin D, and renin. As a possible model system for (anti-Mo-MSV/MLV complex (Mo-MSV = Moloney murine sarcoma virus; MLV = murine leukemia virus)) activity was investigated. Both compounds were found to inhibit moderately the focus formation of Mo-MSV/MLV complex in NIH3T3 cells (compound 4, IC50 = 1.8 microM; compound 6, IC50 = 1.0 microM).

Amino Acid Sequence↗
PubMed1994

Electron microscopic studies on the inhibition of degranulation of rat mast cells by a novel anti-allergic agent, PTPC.

When rat mast cells sensitized by IgE antibody were exposed to antigen, transmission electron microscopy revealed alteration of the granules, cavity formation by fusion of the perigranular membrane and granule release by the fusion of the cavity membrane with the mast cell membrane. Scanning electron microscopy disclosed the extrusion of smooth and round bodies from pores formed on the cell surface. These changes were accompanied by the release of histamine. The inhibition of this degranulation by a novel anti-allergic agent, 6-(1-pyrrolidinyl)-N-(1H-tetrazol-5-yl)-2-pyrazinecarboxamide (PTPC), was evaluated quantitatively as an inhibition of the granule alteration and cavity formation. At a concentration of 100 nM, PTPC inhibited the granule alteration and cavity formation as well as histamine release. In the same concentration, PTPC significantly increased the cyclic AMP content in the mast cells. These results suggest that the inhibition of the morphological changes in mast cells by PTPC might be due to the increased cyclic AMP caused by the agent and plays an important role in the suppression of chemical mediators release.

Animals↗
PubMed1994

Egg-jelly signal molecules for triggering the acrosome reaction in starfish spermatozoa.

It was in the early 1950s that J.C. Dan discovered the acrosome reaction in sea urchins, starfishes and several other marine invertebrates at Misaki Marine Biological Station on the Pacific coast of Japan. We now know that in many animals including mammals the acrosome reaction is an essential, and probably the most central, change in spermatozoa for fertilization. Starfish spermatozoa undergo the acrosome reaction upon encountering the jelly coat consisting of glycoproteins, steroid saponins, oligopeptides and inorganic components. To induce the acrosome reaction, three egg jelly components act in concert on the spermatozoa: a highly sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of sulfated steroidal saponins named Co-ARIS, and a group of glutamine-rich tetratriacontapeptides named sperm activating peptide (SAP). The action of ARIS is quite species-specific due to the specificity of ARIS-receptors in a restricted domain of the sperm surface and depends very much on sulfated saccharide chains. Co-ARIS is not much species-specific and its action depends on the sulfate group and steroid side chain. SAPs have a ring of 25 residues and increase the intracellular pH of spermatozoa. None of them can induce the acrosome reaction by itself in normal sea water, but ARIS does induce it in high Ca2+ or high pH sea water. Although a combination of ARIS and either Co-ARIS or SAP induces the acrosome reaction in normal sea water, all three are required to mimic the full activity of dissolved jelly coat.

Acrosome↗
PubMed1993

Studies of HIV-1 protease inhibitors. I. Incorporation of a reduced peptide, simple aminoalcohol, and statine analog at the scissile site of substrate sequences.

Inhibitors of the protease of human immunodeficiency virus type-1 (HIV-1) were designed and synthesized. A reduced peptide, simple aminoalcohol, and statine analog, 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), were inserted at the scissile site of substrate sequences of HIV-1 protease. While both reduced peptides and simple aminoalcohol derivatives were weak inhibitors, the peptides containing AHPPA demonstrated moderate inhibitory activity. The more potent alcohol configuration of AHPPA is (R), which is opposite to the configuration in potent inhibitors of other aspartic proteases. In particular, compound 28 ((3R,4S)-4-(N-tert-butoxycarbonyl- L-glutaminyl-L-asparaginyl)amino-3-hydroxy-5-phenylpentanoic acid 2'-methylbutylamide) had a Ki of 0.36 microM and exhibited excellent enzyme specificity.

Amino Acid Sequence↗
PubMed1993

Studies of HIV-1 protease inhibitors. II. Incorporation of four types of hydroxyethylene dipeptide isosteres at the scissile site of substrate sequences.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors containing four types of hydroxyethylene dipeptide isosteres were designed and synthesized. These inhibitors consist of eight stereoisomers of phenylalanylproline (Phe-psi[H.E.]-Pro), four stereoisomers of phenylalanylalanine [Phe-psi[H.E.]-Ala), and one stereoisomer each of phenylalanylglycine (Phe-psi[H.E.]-Gly) and cyclohexylalanylalanine (Cha-psi[H.E.]-Ala) hydroxyethylene dipeptide isosteres. For the synthesis of the latter two isosteres, a newly developed synthetic method for gamma-lactone was applied. The inhibitory activities of these peptides were evaluated by cleavage assay of partially purified gag proteins or purified synthetic peptide. Of the inhibitors examined, compounds 2c (Z-Asn-(2S,3R,4S,5S)-Phe-psi[H.E.]-Pro-NHB(un); Bu(n) = n-butyl, Ki = 0.50 microM), 21a (Z-Asn-(2R,4S,5S)-Phe-psi[H.E.]-Ala- NHBu(n), Ki = 0.34 microM) and 23 (Z-Asn-(2R,4S,5S)-Cha-psi[H.E.]-Ala- NHBu(n), Ki = 0.46 microM) were moderately potent inhibitors. The results revealed that the alkyl substituent at C2 is essential, and the stereochemistry of the hydroxyethylene dipeptide isosteres greatly affected their inhibitory activities.

Amino Acid Sequence↗
PubMed1992

Mitochondrial encephalomyopathies with the mutation of the mitochondrial tRNA(Leu(UUR)) gene.

Four families with mitochondrial encephalomyopathy are described. Probands of three families had typical clinical presentations of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), but the proband of family 4 lacked strokelike episodes. The mitochondrial DNA mutation of tRNA(Leu(UUR)) (transfer ribonucleic acid specific to leucine (UUR codon)) found in MELAS was examined in muscle DNA obtained from biopsy samples of the probands of four families and the maternal relatives of family 2. The mutation was detected in all muscle samples, and the degree of the mutated DNA was 68% to 84% by Southern blot analysis. However, the clinical patterns of the maternal relatives of family 2 were mild and distinctly different from MELAS. The same mutation was also detected in blood-derived DNA samples of all family members examined, including healthy mothers but not fathers, although the degree of mutation did not correlate with the clinical severity. These results confirmed the maternal inheritance of this disease and suggested that the mitochondrial DNA mutation (tRNA(Leu(UUR))) may cause clinical symptoms other than MELAS. The clinical findings of mitochondrial encephalomyopathy should be reinvestigated in terms of the mitochondrial gene mutation; the polymerase chain reaction method will be useful for screening for this mutation of mitochondrial DNA in blood samples.

Acidosis, Lactic↗
PubMed1990

The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites.

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

Animals↗
PubMed1990

Electron microscopic radioautographic study of the localization of an anti-allergic agent, Tranilast, in rat mast cells.

We have previously reported that Tranilast, an anti-allergic agent, was rapidly taken into the cytoplasm of rat mast cells in vitro by means of light microscopic radioautography. The present study was performed at the electron microscopic level to elucidate the fine localization of this agent in the mast cells. The results revealed that the number of radioautographic silver grains in the cells increased by the incubation with 3H-labelled Tranilast for 0 to 60 min. and that many silver grains were localized on the specific granules, especially on the perigranular membranes. These results suggest that the mode of inhibitory action of mast cell degranulation by Tranilast is related to the specific localization of this agent on the perigranular membranes.

Animals↗