Biomedical subjects

R Nagata

Publications and source records attributed to R Nagata.

At least 19 recordsLinked to original sources

PubMed2007

CYP2C76-mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.

Animals↗

PubMed2005

A national surveillance of Shiga toxin-producing Escherichia coli in food-producing animals in Japan.

To assess the public health risk, the prevalence and anti-microbial resistance of Shiga toxin-producing Escherichia coli (STEC) among food-producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non-motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae-gamma1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae-beta1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae-positive isolates were not observed. Bovine isolates showed less anti-microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi-resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.

Animals↗

PubMed2001

Oxindole derivatives as orally active potent growth hormone secretagogues.

A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone (GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity (EC(50) = 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at 10 mg/kg twice a day. Compound 37S displaced the binding of (35)S-MK-677 to human GHS-R with an IC(50) value of 1.2 +/- 0.2 nM.

Administration, Oral↗

PubMed2001

Synthesis of tricyclic indole-2-carboxylic [correction of caboxylic] acids as potent NMDA-glycine antagonists.

The practical synthesis of a series of tricyclic indole-2-carboxylic acids, 7-chloro-3-arylaminocarbonylmethyl-1,3,4,5-tetrahydrobenz[cd]indole-2-carboxylic acids, as a new class of potent NMDA-glycine antagonists is described. The synthetic route to the key intermediate 12a comprises a regioselective iodination of 4-chloro-2-nitrotoluene, modified Reissert indole synthesis, Jeffery's Heck-type reaction with allyl alcohol, Wittig-Horner-Emmons reaction, and iodination at the indole C-3 position. The key step in the route is an intramolecular cyclization of 12a to give the tricyclic indole structure. Two methods of cyclization, (1) an intramolecular radical cyclization of 12a and (2) a sequence of intramolecular Heck reaction of 12a followed by a 1,4-reduction, were performed. The resulting tricyclic indole diester 13a was selectively hydrolyzed to afford the desired tricyclic indole monocarboxylic acid 16 on a multihundred gram scale without any chromatographic purifications. Optical resolution of 16 to (-)-isomer 17 and (+)-isomer 18 was carried out, and the resulting isomers were derivatized, respectively. Evaluation of the optically active derivatives for affinity to the NMDA-glycine binding site using the radio ligand binding assay with [(3)H]-5,7-dichlorokynurenic acid revealed that the derivatives of (-)-isomer 17 were more potent than the others and that especially substituted anilide (-)-isomer 24 (K(i) = 0.8 nM) showed high affinity.

Animals↗

PubMed2001

Experimental thromboembolic stroke in cynomolgus monkey.

To develop an experimental model of thromboembolic stroke without intracranial surgery, an autologous blood clot was delivered to the middle cerebral artery (MCA) via the internal carotid artery in cynomolgus monkeys. Male cynomolgus monkeys, in which a chronic catheter had been earlier implanted in the left internal carotid artery, were used. The clot was flushed into the internal carotid artery under sevofluorane anesthesia. A neurologic deficit score was assigned after MCA embolization. After 24 h, cerebral infarct size and location were determined by the TTC staining method. Cerebral blood flow (CBF) was measured prior to and after MCA embolization, using positron emission tomography (PET). After embolization, long-lasting and profound extensor hypotonia of the contralateral upper and lower limbs, and mild to severe incoordination were observed. Contralateral hemiplegia was observed over the following 24 h. In gross morphologic observation of the brain, the lesions involved mostly the caudate nucleus, putamen, globus pallidus and insular cortex. CBF was maximally reduced in the left MCA territory, but not in the right MCA territory. This model is relevant to thromboembolic stroke in human in neurologic dysfunction and histopathologic brain damage.

Animals↗

PubMed2001

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi.

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.

Amino Acid Sequence↗

PubMed2001

Pharmacological profile of a new orally active growth hormone secretagogue, SM-130686.

SM-130686, an oxindole derivative, is a novel orally active GH secretagogue (GHS) which is structurally distinct from previously reported GHSs such as MK-677, NN703 and hexarelin. SM-130686 stimulates GH release from cultured rat pituitary cells in a dose-dependent manner. Half-maximum stimulation was observed at a concentration of 6.3+/-3.4 nM. SM-130686-induced GH release was inhibited by a GHS antagonist, but not by a GH-releasing hormone antagonist. SM-130686 dose-dependently inhibited the binding of radiolabeled ligand, (35)S-MK-677, to human GHS receptor 1a (IC(50)=1.2 nM). This indicates that SM-130686 stimulates GH release through the GHS receptor. The effect of a single oral administration of SM-130686 on GH release in pentobarbital-anesthetized rats was studied. After treatment with 10 mg/kg SM-130686, plasma GH concentrations measured by radioimmunoassay significantly increased, reaching a peak at 20-45 min, and remained above baseline during the experimental period (60 min). The anabolic effect of repetitive SM-130686 administration was studied in rats. Rats received 10 mg/kg SM-130686 orally twice a day and were weighed every day for 9 days. At day 9 there was a significant increase in both the body weight and the fat free mass (19.5+/-2.1 and 18.1+/-7.5 g respectively). Serum IGF-I concentration was also significantly elevated 6 h after the last dose of SM-130686. An endogenous GHS ligand for the GHS receptor has recently been identified from stomach extract and designated as ghrelin. The GH-releasing activity in vitro relative to ghrelin (100%) was about 52% for SM-130686. It is likely that SM-130686 is a partial agonist for the GHS receptor. In summary, we describe here an orally active GHS, SM-130686, which acts through the GHS receptor. Repetitive administration of SM-130686 to rats, similar to repetitive administration of GH, significantly increased the fat free mass by an amount almost equal to the gain in body weight.

Administration, Oral↗

PubMed2000

Effects of ropinirole on motor behavior in MPTP-treated common marmosets.

The effects of ropinirole (4-[2-(dipropylamino)ethyl]-2-indolinone monohydrochloride), a nonergoline dopamine receptor agonist with a high affinity for native dopamine D(2)-like receptors, on Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 2.5 mg/animal in common marmosets were examined and compared to the effects of bromocriptine. Ropinirole (0.1-3 mg/kg, PO) increased motor activity dose dependently and reversed akinesia or uncoordinated movement in MPTP-treated marmosets. The activities for ropinirole were very similar to those of bromocriptine. Ropinirole had, however, several properties that differed from those of bromocriptine. Ropinirole caused a more rapid onset of anti-Parkinsonian activity compared to bromocriptine, and had a potency more than five times greater than that of bromocriptine in the improvement of motor deficits. The combination of ropinirole and L-DOPA increased the effectiveness of ropinirole or L-DOPA alone, and produced a more marked additive effect on motor activity than did bromocriptine and L-DOPA. Chronic administration of ropinirole for 21 days produced a statistically significant increase in motor activity compared to the initial administration, and akinesia scores, measured through rating the quality of movements, were also improved without obvious dyskinesia. This study suggests that ropinirole is a dopamine D(2)-like receptor agonistic drug of potential use in the treatment of Parkinson's disease.

Animals↗

PubMed2000

Effects of ropinirole on various parkinsonian models in mice, rats, and cynomolgus monkeys.

Ropinirole (4-[2-(dipropylamino)ethyl]-2-indolinone monohydrochloride) a nonergoline dopamine receptor agonist with high affinity for native dopamine D(2)-like receptors in human caudate tissue, was tested with respect to the stimulation of postsynaptic brain dopamine receptors in standard preclinical models of Parkinson's disease. Additionally, in these animal models the antiparkinsonian activity of ropinirole was compared to that of bromocriptine. The ED(50)s (95% confidence limits) of ropinirole and bromocriptine on the turning behavior in 6-OHDA-lesioned rats were 20.17 mg/kg (14.27-26.88 mg/kg) and 11.99 mg/kg (9.37-14.17 mg/kg), respectively. The ED(50)s (95% confidence limits) of ropinirole and bromocriptine on the catalepsy induced by reserpine were 18.55 mg/kg (15.29-22.99 mg/kg) and 12.56 mg/kg (10.25-14.64 mg/kg), respectively. Ropinirole and bromocriptine had no effect on the tremors induced by oxotremorine in mice, whereas atropine markedly suppressed the tremors. The ED(50)s (95% confidence limits) of ropinirole and bromocriptine on the tremors in VMT-lesioned monkeys were 0.18 mg/kg (0.12-0.29 mg/kg) and 2.63 mg/kg (1.06-6.45 mg/kg), respectively. In rodent parkinsonian models, bromocriptine was more potent than ropinirole; however, in the nonhuman primate parkinsonian model, ropinirole was a more potent inhibitor of parkinsonian activity than bromocriptine. This study suggests that ropinirole is a dopamine D(2)-like receptor agonistic drug of potential use in the treatment of Parkinson's disease.

Animals↗

PubMed2000

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.

Amyloid↗

PubMed2000

East-West development: understanding the usability and acceptance of foreign data in Japan.

The introduction in Japan of New GCP regulations, as well as the internationalization of clinical trials under the ICH process (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use), has meant that the system is now faced with a number of serious challenges. This is shown by the dramatic decrease in registration of new drugs. Identifying the actors in the new context is essential. We shall first define precisely what is the Regulatory Authority in Japan, and which are the Regulatory Requirements to be fulfilled. We shall then describe the new process for evaluation of foreign clinical data. One of the major issues will be to determine in which cases a complete clinical data package will be sufficient to support regulatory submission, and in which cases additional clinical studies or bridging studies, and what kind of bridging studies will be required.

Asian People↗

PubMed1999

Conformation of the primary binding loop folded through an intramolecular interaction contributes to the strong chymotrypsin inhibitory activity of the chymotrypsin inhibitor from Erythrina variegata seeds.

We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (Ki, 5.6 x 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (Ki, 1.3 x 10(-6) M) than ECI (Ki, 9.8 x 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (Ki, 5.7 x 10(-7) M) than ETIa (Ki, 1.3 x 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

Alanine↗

PubMed1999

[Molecular identification of cytokinin-specific binding protein].

Synthetic phenylurea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we found the presence of a cytokinin-specific binding protein (CSBP) with high affinity for 4PU30 (Ka for 4PU30 = 4 x 10(10) M-1) in the soluble fraction of etiolated mung bean seedlings. We purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel ligated with 4PU. Analysis of its cDNA revealed that CSBP was a novel member of a major pollen allergen/pathogenesis-related protein family with a calculated molecular weight of 17 kDa. Recombinant CSBP was expressed in Escherichia coli was confirmed to bind specifically to cytokinins.

Amino Acid Sequence↗

PubMed1998

Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata).

Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and functional analysis of cynomolgus monkey CYP1A2.

Complementary DNA fragments encoding cynomolgus monkey CYP1A2 were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) method from the liver total RNA of a 3-methylcholanthrene (3-MC)-treated cynomolgus monkey. The nucleotide sequence determined was 1630 bp long and contained an open reading frame for a polypeptide of 516 residues. The nucleotide and the deduced amino acid sequences of cynomolgus monkey CYP1A2 showed 95.1 and 92.8% identities to those of human CYP1A2, respectively. The level of CYP1A2 mRNA in the liver of untreated cynomolgus monkey was very low. Treatment with 3-MC increased it. Still, it was one-fortieth that of CYP1A1. Cynomolgus monkey CYP1A2 expressed in recombinant yeasts activated 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8dimethylimidazo[4,5-flquinoxaline (MeIQx) at efficient rates in the umu mutagenicity test. This cytochrome P450 (CYP) also activated 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but less efficiently. These results indicate that cynomolgus monkeys have a functionally active CYPIA2 gene, but its expression level is very low in the liver of untreated cynomolgus monkeys.

Amino Acid Sequence↗

PubMed1998

Dysregulation of trace element composition in ovariectomized cynomolgus monkey bones.

One of the challenging issues in modern biomedical science is the increasing number of osteoporosis patients due to the expansion of elderly populations. Among aging-related pathogenic changes, alterations in bone function and skeletal pathogenesis is a particularly important issue of concern. Osteoporosis is one of the most serious bone-related pathogenic states, as it causes serious loss of quality of life. Alterations in estrogen levels in accordance with aging are one of the key risk factors for osteoporosis. Complexed estrogen actions on bones can be traced by analyzing bone mineral components, as those elements accumulate as mineral complexes, reflecting the context of multiple cellular reactions such as bone resorption/osteogenesis. We have analyzed bone trace element composition in ovariectomized (OVX-treated) Cynomolgus monkey models in this study. In order to gain insights into the effects of such defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in vertebral bones of OVX-treated Cynomolgus monkeys. An assessment of these trace element spectra in OVX model animals is discussed. These results could provide useful markers for understanding the physiological states of bones in postmenopausal women.

Animals↗

PubMed1997

Marmoset liver cytochrome P450s: study for expression and molecular cloning of their cDNAs.

Northern blot and immunoblot analyses indicated that considerable levels of CYP2B, CYP2C, CYP2D, CYP2E, and CYP3A were expressed in the liver of untreated marmosets. CYP1A was also expressed but to lesser extents. CYP3A mRNA was also detectable in the small intestine of untreated marmoset; the amount was increased by treatment with polychlorinated biphenyl. From a liver cDNA library, two cDNA clones coding for CYP2D19 and CYP3A21 (clones CM2D-1 and CM3A-10, respectively) were isolated. CM2D-1 and CM3A-10 contained an entire coding region for polypeptide 497 and 503 amino acid residues, respectively. The deduced amino acid sequences of CYP2D19 and CYP3A21 showed 90% identities to human CYP2D6 and CYP3A4, respectively. The value of CYP3A21 was 3% lower than that of cynomolgus monkey CYP3A8. On the other hand, these values were 11 to 23% higher than those of the other experimental animals, including dogs, rabbits, guinea pigs, rats, mice, and hamsters. These results indicate that the marmoset stands at a midpoint between human and nonprimate experimental animals.

Amino Acid Sequence↗

PubMed1997

A monoclonal antibody to astrocytes, subepithelial fibroblasts of small intestinal villi and interstitial cells of the myenteric plexus layer.

A monoclonal antibody was developed, using cultured subepithelial fibroblasts of rat duodenal villi as the antigenic material, by in vitro immunization. Hybridomas were selected on cryosections of rat brain and small intestine using indirect immunofluorescence techniques. The monoclonal antibody, termed 8E1, was very useful to label GFAP-positive astrocytes in the adult rat brain, subepithelial fibroblasts of intestinal villi and Thuneberg's fibroblast-like interstitial cells associated with the myenteric plexus layer. These cells are known to be stellate in shape, forming a cellular network with extended cell processes that communicate via gap junctions. Immature astrocytes such as radial fibers were not immunolabeled with mAb-8E1. Immunoelectron microscopy revealed clustered gold labeling of 8E1 antigen on the intermediate filaments of cultured astrocytes and cultured subepithelial fibroblasts. This staining pattern was different from the continuous and linear gold labeling of GFAP and vimentin. MAb-8E1 did not immunoblot purified human brain GFAP nor bovine lens vimentin, and so was not neutralized by preabsorption with these molecules. Immunoblot analysis after SDS-PAGE showed that the antigen recognized by mAb-8E1 was a Mr 50K protein in the rat cerebra, and a Mr 56K protein in the cultured subepithelial fibroblasts. This study showed that astrocytes and some types of stellate cells share distinct antigenic properties in the components of their intermediate filaments.

Animals↗