Biomedical subjects

M Miyagi

Publications and source records attributed to M Miyagi.

At least 55 records · Page 3Linked to original sources

PubMed1998

Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity.

Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.

Amino Acid Sequence↗

PubMed1998

A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides.

We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.

Amino Acid Sequence↗

PubMed1998

Renal ACE immunohistochemical localization in NIDDM patients with nephropathy.

A role of renal angiotensin-converting enzyme (ACE) in diabetic nephropathy has been suggested. Immunohistochemical localization of ACE was studied in 20 non-insulin-dependent diabetes mellitus patients with diabetic nephropathy and 17 healthy kidney transplant donors, with ACE gene insertion/deletion (I/D) polymorphism also examined in the latter. Immunohistochemical studies indicated that ACE staining was significantly (P < 0.01) enhanced in glomeruli and slightly decreased in proximal tubules in diabetic patients. Glomeruli positive for ACE immunostaining were observed in 23.5% of the healthy subjects and in 80% of the diabetic patients. All patients with nodular lesions had ACE-positive glomeruli and showed significantly (P < 0.01) more intense glomerular ACE immunostaining than patients without nodular lesions. Among healthy controls, subjects with the DD genotype had ACE-positive glomeruli more frequently and tended to show slightly increased intensity on proximal tubule ACE immunostaining compared with subjects with other genotypes. These observations suggest that increased ACE localization in glomeruli is likely to be one of the factors in the increased renin-angiotensin system activity in glomeruli in patients with diabetic nephropathy. There is a possibility that ACE gene I/D polymorphism may be related to renal ACE immunohistochemical localization.

Diabetes Mellitus, Type 2↗

PubMed1998

Identification of the catalytic triad residues of porcine liver acylamino acid-releasing enzyme.

Acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] is a tetrameric serine protease, which belongs to the oligopeptidase family and specifically removes acetyl amino acids from N-terminally acetylated peptides. By using diisopropyl fluorophosphate, we previously identified one of the residues comprising the catalytic triad of this enzyme as Ser587 [Miyagi, M. et al. (1995) J. Biochem. 118, 771-779]. To elucidate the other two residues forming the catalytic triad of this new serine-type protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with other oligopeptidases was substituted by site-directed mutagenesis, were expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme. All of the recombinant AAREs were estimated to have similar conformational and quaternary structures to the native porcine liver enzyme from their CD spectra and behavior on gel-filtration, but the mutants in which Ala587, Asn675, or Tyr707 was substituted for Ser587, Asp675, or His707, respectively, did not show detectable hydrolytic activity toward acetyl-L-methionyl L-alanine. These facts suggest that Ser587, Asp675, and His707 are essential residues for the AARE activity and comprise the catalytic triad of the enzyme in this order. Thus, AARE has been shown to have a protease-like domain in its C-terminal region, as do other proteins classified as members of the oligopeptidase family.

Amino Acid Sequence↗

PubMed1998

In vitro release of tranilast from oily gels and penetration of the drug into Yucatan micropig skin.

For the transdermal delivery of tranilast (TL), a drug used for the treatment of skin diseases such as keloids and hypertrophic scars, its oily gels were prepared; its in vitro release and penetration into Yucatan micropig skin were evaluated. In the gels that consisted of hydrogenated soybean phospholipids (HSL) and octyl isononanoate (IOIN), a fatty-acid ester, the release of TL from the gels was proportional to the drug content, and the extent of TL released up to 6 h from them was approximately 70% of the amount of applied TL. On the other hand, with the gels consisting of HSL and isocetyl isostearate (ICIS), the release of TL from the gels was about half of that from IOIN gels, even at the same drug concentration. When oily gels were used, the TL skin concentration was rapidly increased compared with the level obtained with suspensions. With 0.1% IOIN gel, a high concentration of TL (ca. 160 microg/g) in the dermis was obtained and continued until at least 48 h. These results suggest that oily gels may be useful for the topical application of TL.

Animals↗

PubMed1998

Formation of water-insoluble gel in dry-coated tablets for the controlled release of theophylline.

Sodium alginate (ALNa) of a natural polysaccharide is known to form a water-insoluble gel when combined with a bivalent metal. In this study, we prepared tablets containing ALNa and calcium gluconate (GLCa) as a bivalent metal, and studied the application of the water-insoluble gel involving the controlled release of a test drug by permeation of water. Dry-coated tablets containing theophylline (TP) as a model drug, ALNa and GLCa were prepared by the dry power compression method. The controlled release of TP was evaluated by the dissolution test according to JP XIII. The release rate was extremely high for the tablets which contained only TP and GLCa. A zero order or sigmoidal release profile was observed for the tablets that contained only TP and ALNa. On the other hand, the lowest dissolution rate and a sigmoidal release profile were observed for the tablet containing TP and GLCa in its core and ALNa in its outer phase. These results suggest that dry-coated tablets containing ALNa and GLCa and prepared by the direct powder compression method would be useful for the controlled release of drugs.

Alginates↗

PubMed1998

Low-dose basic fibroblast growth factor and vascular endothelial growth factor for angiogenesis in canine acute hindlimb insufficiency.

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have shown strong angiogenetic effects in ischemic animals; however, whether such a beneficial effect could be achieved using low doses remains to be determined. The effects of identical low-level doses of these substances on the creation of collateral circulation in canine acute hind limb insufficiency were evaluated. Anesthetized dogs that had undergone left femoral artery occlusion received 20 microg (2 microg/kg) intravenous boluses of either bFGF or VEGF 3 times at 2-day intervals for the first week only, animals on vehicle saline injection served as controls. All groups, control (n=8), bFGF-treated (n=8), and VEGF-treated (n=6) underwent angiography, blood flow measurement (in ml/min) on the day of ligation (day 0), and at 7, 14 and 28 days, then underwent ischemic limb muscle biopsy at 28 days. Angiogenic-treated groups showed remarkable enhanced collateral circulation at 7 days, which was maintained up to 28 days, and the main collateral source artery of the angiogenic-treated groups dilated by 14 days. Many neovascularized arterioles in specimens of the angiogenic groups were recognized without any tissue edema or necrosis. Even low doses of bFGF or VEGF were enough to augment collateral circulation with no side-effects, and short treatment after acute ischemia was effective. Low-dose bFGF or VEGF may be therapeutical effective options in patients with acute lower limb vascular disease.

Animals↗

PubMed1998

Osteopathy in broiler chicks fed toxic mimosine in Leucaena leucocephala.

Further studies of mimosine toxicity in broiler chicks were done to clarify a possibility of osteopathy. The mineral content and density of femur and the strength, ductility, and toughness for the index of mechanical properties significantly decreased in the 1% mimosine group, compared with those in the control and restricted groups. The stiffness had a decreasing tendency in the 1% mimosine group. Consequently, it was concluded that chicks fed ad libitum a 1% mimosine diet for 12 days developed osteopathy. The bone mineral density and the strength of the restricted group were lower than those of the control group, and those of the 1% mimosine group were still lower than those of the restricted group. Contents of pyridinoline and deoxypyridinoline in the excrement were significantly higher in the restricted group than those in the control group, but the contents in the 1% mimosine group were significantly lowest among the groups. Osteopathy in chicks fed mimosine, therefore, seemed to be done by loss of appetite and changing to a low turnover of bone caused by mimosine.

Amino Acids↗

PubMed1998

Pilot study on the effect of a mouthrinse containing silver zeolite on plaque formation.

A double-blind cross-over study was performed to evaluate the inhibitory activity of silver zeolite (SZ) mouthrinse on plaque formation. Eleven dental students participated in this study. SZ mouthrinse was prepared by suspending SZ powder into phosphate-buffered saline (PBS) at a concentration of 3% (w/w). Type-A zeolite was used as a placebo. Before the experiment, the subjects were rendered plaque-free by professional prophylaxis. They then suspended any oral hygiene for five days, during which time they rinsed with either SZ or type-A zeolite mouthrinse twice a day. SZ significantly reduced plaque formation compared to the placebo (p < 0.05), suggesting that silver ions released from the SZ inhibited plaque formation.

Adult↗

PubMed1997

Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization.

A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of glycyl-L-histidyl-L-lysine (GHK), a liver-cell growth factor isolated from human plasma, and its metabolite, L-histidyl-L-lysine (HK), in rat plasma. Both high selectivity and sensitivity were achieved by the use of solid-phase extraction with a Bond-Elut Certify cartridge, ion-pair chromatography with 1-pentanesulfonate on a 5-microm Capcell Pak C18 UG120 column (250x4.6 mm I.D.) with a guard column, and by post-column derivatization with o-phthalaldehyde (OPA). GHK and HK were extracted from 0.1 ml of rat plasma after addition of o-phenanthroline to protect against degradation. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly.

Animals↗

PubMed1997

Paracatalytic inactivation of L-2-haloacid dehalogenase from Pseudomonas sp. YL by hydroxylamine. Evidence for the formation of an ester intermediate.

Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp. YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K. (1995) J. Biol. Chem. 270, 18309-18312). We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate. Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme. To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments. The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da. Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10. When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher. Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.

Acetates↗

PubMed1997

In vivo intravascular laser photodynamic therapy in rabbit atherosclerotic lesions using a lateral direction fiber.

BACKGROUND AND OBJECTIVE: This study was performed to evaluate the possibility of inducing regression of atherosclerotic foci by photodynamic therapy (PDT) using hematoporphyrin derivative (HpD). STUDY DESIGN/MATERIALS AND METHODS: Atherosclerotic rabbits were divided into four groups: A (n = 6) and C (n = 6) were given 5 mg/kg of HpD intravenously; Groups B (n = 4) and D (n = 4) were not. Twenty-four hours after HpD administration, the aortae of groups A and B were exposed to 200 mw output argon dye laser beam at 630 nm for 10 minutes; groups C and D were exposed to 400 mw for 5 minutes. Three rabbits from groups A and C and two rabbits from groups B and D were sacrificed immediately after laser photoradiation, being named groups A 0, C 0 and groups B 0, D 0, respectively. Groups A 7, C 7, and Groups B 7, D 7 were sacrificed 7 days after the photoradiation. RESULTS: In groups A 7 and C 7, most intimal cells and endothelial cells had become necrotic and disappeared, and a loss of intima was observed. No such changes were found in groups B 7, D 7. CONCLUSION: The above data suggest that PDT caused effective regression of the atherosclerotic lesions.

Animals↗

PubMed1997

Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme.

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.

Amino Acid Sequence↗

PubMed1997

Renal angiotensin-converting enzyme localization in diabetic rats and the effect of low protein diet.

Recent evidence suggests a role of angiotensin-converting enzyme (ACE) in diabetic nephropathy. The effect of diabetes and low protein diet on renal immunohistochemical ACE localization was studied in streptozotocin-induced DM rats. Immunohistochemical ACE localization was reduced in DM rats, and a low protein diet partially resolved this abnormality while inhibiting the progression of diabetic nephropathy.

Albuminuria↗

PubMed1996

Amino acid sequence and carbohydrate structure of a recombinant human tissue factor pathway inhibitor expressed in Chinese hamster ovary cells: one N-and two O-linked carbohydrate chains are located between Kunitz domains 2 and 3 and one N-linked carbohydrate chain is in Kunitz domain 2.

Human tissue factor pathway inhibitor is a protease inhibitor with three tandem Kunitz-type inhibitory domains. The recombinant protein (r-hTFPI) was produced using Chinese hamster ovary cells, and its polypeptide and carbohydrate chain structures were analyzed. The complete amino acid sequence, composed of 276 residues, was determined using a protein sequencer after protease digestion and it was identical to that predicted from the cDNA sequence. Among three potential N-glycosylation sites, both Asn117 and Asn167 were fully N-glycosylated but Asn228 was not. Thr175 was also fully O-glycosylated, but Ser174 was partially O-glycosylated. Carbohydrate composition and mass spectrometric analyses of the undecapeptide OG-11 (residues Leu 170approximately Leu180) showed that two O-linked carbohydrate chains consisted of a type-1 core structure (Gal-GalNAc-Ser/Thr) with 0-3 mol of N-acetylneuraminic acid(s). The N-linked carbohydrate chains were analyzed by two-dimensional carbohydrate mapping combined with sequential glycosidase digestion, after the reducing-ends of carbohydrate residues were tagged with 2-aminopyridine and non-reducing-end sialic acids were removed with sialidase. All the N-linked structures in r-hTFPI were complex-type carbohydrate chains with one fucose residue attached to the reducing-end GlcNAc and consisted of bi-, tri-, and tetraantennary carbohydrate chains in the ratio 1.9:1.3:1.0. Fucosylated tri- and tetraantennary carbohydrate chains with one or two N-acetyllactosaminyl repeats were also found (30% of carbohydrate chains determined). Thus, the region between Kunitz domains 2 and 3 encoded by exon 7 was highly glycosylated by two O-linked carbohydrate chains at Ser174 and Thr175 and one N-linked carbohydrate chain at Asn167. These results indicated that the region is occupied by a cluster of three bulky and acidic carbohydrate chains.

Amino Acid Sequence↗

PubMed1996

A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning and tissue localization.

A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.

Agglutinins↗

PubMed1996

Dopamine receptor affinities in vitro and stereotypic activities in vivo of cabergoline in rats.

An ergot alkaloid derivative, cabergoline, and its metabolites were investigated for their affinities for dopamine D1 and D2 receptors in rat striatum in vitro in comparison with those of bromocriptine and pergolide. The affinity for D1 receptors was in the following order: pergolide > des-dimethylaminopropyl cabergoline (FCE21904) > cabergoline > or = bromocriptine > or = des-methyl cabergoline (FCE27395) > or = des-ethylcarbamoyl cabergoline (FCE21590). From the effects of GTP on these affinities for the D1 receptor, cabergoline, some of its metabolites, and pergolide were characterized as agonists in contrast to bromocriptine which was classified as an antagonist. The affinity for D2 receptors was ranked as follows: pergolide > or = cabergoline > or = FCE27395 > or = FCE21904 > bromocriptine > FCE21590 > carboxylic acid-type derivative of cabergoline (FCE21589). The affinity of each compound for the D2 receptor was much higher than that for the D1 receptor. The selectivity of cabergoline for D2 receptor was higher than those of bromocriptine and pergolide. Furthermore, these ergot alkaloids were investigated for eliciting stereotypy after subcutaneous administration to normal rats. Pergolide potently induced stereotypy at doses of 0.5 and 1.0 mg/kg, cabergoline slightly induced it only at a high dose of 2.0 mg/kg, whereas bromocriptine did not induce it at any of the doses tested, 10-40 mg/kg. These results suggest that pharmacological properties of cabergoline for the D1 and D2 receptors differ from those of bromocriptine and pergolide.

Animals↗

PubMed1996

Effect of cabergoline, a long-acting dopamine D2 agonist, on reserpine-treated rodents.

We studied the characterization of cabergoline, a new ergot alkaloid derivative and a selective dopamine D2 receptor agonist, in comparison to bromocriptine and pergolide in reserpine-treated rodents. Cabergoline (0.25-1.0 mg/kg, s.c.) improved dose-dependently the reserpine-induced akinesia that was assessed on the locomotor activity, and the efficacy lasted longer than those of bromocriptine (1.25-5.0 mg/kg, s.c.) or pergolide (0.0625-0.5 mg/kg s.c.). Cabergoline (ED50 = 1.10 mg/kg, at 4 h after the administration of drugs) also reversed catalepsy, the failure to correct an externally imposed posture, and its efficacy was stronger and longer than bromocriptine (ED50 = 4.65 mg/kg, at 4 h). Further, reserpine-induced rigidity was improved equally by cabergoline (0.125-1.0 mg/kg, i.v) and bromocriptine (1.0 mg/kg, i.v.). When cabergoline was administered together with 3(3,4-dihydroxyphenyl)-L-alanine (L-DOPA), the effects were additive. Our results indicate that the long-lasting effects of cabergoline could be beneficial for treating Parkinson's disease.

Animals↗