Biomedical subjects

Takashi Inui

Publications and source records attributed to Takashi Inui.

10 recordsLinked to original sources

PubMed2006

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase.

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.

Amino Acid Sequence↗

PubMed2006

MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration.

We have previously shown that matrix metalloproteinases (MMPs) play a role in osteoclastic bone resorption by facilitating migration of osteoclastic cells toward bone surface through matrices. Of MMPs identified so far, MMP-9 is likely the most important proteinase for the action, since osteoclasts express this enzyme at a tremendously high level. However, no direct evidence has been provided to demonstrate its contribution to bone resorption. In this study, to address this point, we used an MMP-9 antisense phosphothiorate oligodeoxynucleotide (S-ODN), which was shown to inhibit the protein synthesis of MMP-9 efficiently. We demonstrated that the antisense S-ODN inhibited osteoclastic pit formation on matrigel-coated dentine slices in a concentration-dependent manner with a maximum reduction of total pit volume by 53% at 10 microM. These results, taken together, suggest that MMP-9 is involved in osteoclastic bone resorption process possibly by facilitating migration of osteoclasts through proteoglican-rich matrices.

Animals↗

PubMed2006

Prostaglandin D2-mediated microglia/astrocyte interaction enhances astrogliosis and demyelination in twitcher.

Prostaglandin (PG) D2 is well known as a mediator of inflammation. Hematopoietic PGD synthase (HPGDS) is responsible for the production of PGD2 involved in inflammatory responses. Microglial activation and astrogliosis are commonly observed during neuroinflammation, including that which occurs during demyelination. Using the genetic demyelination mouse twitcher, a model of human Krabbe's disease, we discovered that activated microglia expressed HPGDS and activated astrocytes expressed the DP1 receptor for PGD2 in the brain of these mice. Cultured microglia actively produced PGD2 by the action of HPGDS. Cultured astrocytes expressed two types of PGD2 receptor, DP1 and DP2, and showed enhanced GFAP production after stimulation of either receptor with its respective agonist. These results suggest that PGD2 plays an important role in microglia/astrocyte interaction. We demonstrated that the blockade of the HPGDS/PGD2/DP signaling pathway using HPGDS- or DP1-null twitcher mice, and twitcher mice treated with an HPGDS inhibitor, HQL-79 (4-benzhydryloxy-1-[3-(1H-tetrazol-5-yl)-propyl]piperidine), resulted in remarkable suppression of astrogliosis and demyelination, as well as a reduction in twitching and spasticity. Furthermore, we found that the degree of oligodendroglial apoptosis was also reduced in HPGDS-null and HQL-79-treated twitcher mice. These results suggest that PGD2 is the key neuroinflammatory molecule that heightens the pathological response to demyelination in twitcher mice.

Animals↗

PubMed2006

Zebrafish and chicken lipocalin-type prostaglandin D synthase homologues: Conservation of mammalian gene structure and binding ability for lipophilic molecules, and difference in expression profile and enzyme activity.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a bifunctional protein possessing both the ability to synthesize PGD(2) and to serve as a carrier protein for lipophilic molecules. L-PGDS has been extensively studied in mammalian species, whereas little is known about non-mammalian forms. Here, we identified and characterized the L-PGDS homologues from non-mammals such as zebrafish and chicken. Phylogenetic analysis revealed that L-PGDSs of mammalian and non-mammalian organisms form a "L-PGDS sub-family" that has been evolutionally separated from other lipocalin gene family proteins. The genes for zebrafish and chicken L-PGDS homologues consisted of 6 exons, and all of the exon/intron boundaries were completely identical to those of mammalian L-PGDS genes. Zebrafish and chicken L-PGDS genes were clustered with several lipocalin genes in the chromosome, as in the case of mouse and human genes. Gene expression profiles were different among chicken, mouse, human, except for conservation of abundant expression in the brain and heart. The chicken L-PGDS homologue carried weak PGDS activity, whereas the zebrafish protein did not show any of the activity. However, when the amino-terminal region of the zebrafish L-PGDS homologue was exchanged for that of mouse L-PGDS carrying the Cys residue essential for PGDS activity, this chimeric protein showed weak PGDS activity. Both zebrafish and chicken L-PGDS homologues bound thyroxine and all-trans retinoic acid, like mammalian L-PGDSs and other lipocalin gene family proteins. These results indicate that non-mammalian and mammalian L-PGDS genes evolved from the same ancestral gene and that the non-mammalian L-PGDS homologue was the primordial form of L-PGDS but whose major function was and is to serve as a carrier protein for lipophilic molecules. During molecular evolution, the mammalian L-PGDS protein might have acquired effective PGDS activity through substitution of several amino acid residues, especially in the amino-terminal region including the Cys residue, which is essential for PGDS activity.

Amino Acid Sequence↗

PubMed2005

Further application of a two-step heparin affinity chromatography method using divalent cations as eluents: purification and identification of membrane-bound heparin binding proteins from the mitochondrial fraction of HL-60 cells.

Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg(2+) in the first and increasing concentrations of Ca(2+) in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.

Amino Acid Sequence↗

PubMed2005

Structural and mutational analysis of Trypanosoma brucei prostaglandin H2 reductase provides insight into the catalytic mechanism of aldo-ketoreductases.

Trypanosoma brucei prostaglandin F2alpha synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H2 to PGF2alpha in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS.NADP+.citrate at 2.1 angstroms resolution. TbPGFS adopts a parallel (alpha/beta)8-barrel fold lacking the protrudent loops and possesses a hydrophobic core active site that contains a catalytic tetrad of tyrosine, lysine, histidine, and aspartate, which is highly conserved among AKRs. Site-directed mutagenesis of the catalytic tetrad residues revealed that a dyad of Lys77 and His110, and a triad of Tyr52, Lys77, and His110 are essential for the reduction of PGH2 and 9,10-PQ, respectively. Structural and kinetic analysis revealed that His110, acts as the general acid catalyst for PGH2 reduction and that Lys77 facilitates His110 protonation through a water molecule, while exerting an electrostatic repulsion against His110 that maintains the spatial arrangement which allows the formation of a hydrogen bond between His110 and C11 that carbonyl of PGH2. We also show Tyr52 acts as the general acid catalyst for 9,10-PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH2 reductase but also provide an insight into the catalytic specificity of AKRs.

Amino Acid Sequence↗

PubMed2005

Therapeutic effects of ecabet sodium, an antiulcer drug, on dextran sodium sulfate-induced ulcerative colitis in rats.

Ecabet, an antiulcer drug, is reported to be effective in patients with ulcerative colitis. We investigated the effect of ecabet enema on ulcerative colitis in rats and some mechanisms underlying this effect. In vivo ecabet enema showed a therapeutic effect in the rat ulcerative colitis model induced by dextran sodium sulfate in the drinking water. The amount of ecabet bound to damaged mucosa was higher than that bound to normal mucosa 30 min after intrarectal administration. In vitro ecabet accelerated the restitution of epithelial cells, which was not affected by a TGF-beta antibody. Ecabet inhibited the leukotriene B4 production and 5-lipoxygenase activity in human neutrophils. In conclusion, ecabet enema showed a therapeutic effect in rats with ulcerative colitis. This effect may be attributable to the high binding affinity for damaged mucosa, the acceleration of restitution, and the inhibition of leukotriene B4 production.

Abietanes↗

PubMed2004

Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity.

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.

Amino Acid Sequence↗

PubMed2003

Lipocalin-type prostaglandin D synthase (beta-trace) in cerebrospinal fluid: a useful marker for the diagnosis of normal pressure hydrocephalus.

Lipocalin-type prostaglandin D synthase (PGDS) is considered to be mainly produced in the leptomeninges and secreted into cerebrospinal fluid (CSF). We found PGDS levels in CSF of patients with normal pressure hydrocephalus (NPH) (8.99+/-2.59 microg/ml, mean+/-S.D., n=14) to be significantly lower than levels in a control (15.29+/-5.17, n=14, P<0.0001) and other dementia group (19.14+/-4.34, n=7, P=0.0003). Thus, PGDS level in CSF is a useful marker for the differential diagnosis of NPH from other diseases with dementia.

Aged↗

PubMed2002

Characterization of the unfolding process of lipocalin-type prostaglandin D synthase.

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.

Animals↗