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Biomedical subjects

M Miyagi

Publications and source records attributed to M Miyagi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors.

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.

Amino Acid Sequence↗
PubMed2000

Practical asymmetric synthesis of (S)-MA20565, a wide-spectrum agricultural fungicide.

A practical asymmetric synthesis of a wide-spectrum agricultural fungicide, (S)-MA20565 (1), is described. The convergent synthesis was achieved starting from commercially available 3-(trifluoromethyl)aniline (7) in 44% overall yield through five steps and 2-bromobenzaldehyde (9) in 48% overall yield through four steps, respectively. (S)-O-[1-(3-Trifluoromethylphenyl)ethyl]hydroxylamine (2), a key intermediate of 1, was prepared via ruthenium(II)-catalyzed asymmetric transfer hydrogenation of 1-(3-trifluoromethylphenyl)ethanone (6) followed by chlorination using methanesulfonyl chloride and oxyamination using potassium acetohydroxamate with high level of stereocontrol.

Acetamides↗
PubMed2000

Successful transplantation of a cadaveric kidney with post-infectious glomerulonephritis.

This report describes a successful renal Tx in a patient with chronic renal failure, caused by dysplastic kidneys, who received a cadaveric kidney with post-infectious glomerulonephritis. Sequential renal biopsies were performed at 12 h before Tx, and at 1 h and on days 8 and 58 post-Tx. Post-operative hematuria disappeared on day 9 and proteinuria on day 13. Normal graft function was observed within 1 month, with histologic resolution. Our study suggests that while the donor kidney facilitates deposition of certain immune reactants, this is a host (environmental) problem and when transplanted into a new host (new environment), the problem is no longer sustained.

Adolescent↗
PubMed2000

Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding.

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysis of a tryptic digest of NPR-ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn-linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR-ECD deglycosylated with endoglycosidase F2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of untreated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor expressed in COS-1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.

Amino Acid Sequence↗
PubMed1999

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate.

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.

Esters↗
PubMed1999

Pathological evaluation of steroid withdrawal in pediatric renal transplant recipients.

Protocol biopsies were performed to detect and treat subclinical rejection in eight living related pediatric renal transplant recipients who had been withdrawn from steroids. Low-grade tubulitis (t1) and mononuclear cell interstitial inflammation (i1), typical of borderline rejection and low-grade interstitial fibrosis (ci1), and tubular atrophy (ct1), characteristic of chronic rejection, appeared frequently in protocol biopsies more than 3 yr after steroid withdrawal. In contrast, early-type glomerulitis (g), allograft glomerulopathy (cg), arteriolar hyaline thickening (ah), intimal arteritis (v), and fibrous intimal thickening (cv) were not observed in protocol biopsies after steroid withdrawal. Low-dose pulse therapy (methylprednisolone (MP) 250 or 500 mg/d for 3 d) was administered to five patients for borderline and/or acute grade 1a rejection, as determined by protocol biopsies in the absence of clinical rejection. No oral steroids were administered. Renal function in all patients remained satisfactory, without proteinuria, in follow-up periods ranging from 22 to 68 months after steroid withdrawal. Of the eight patients, three grew to almost normal height (> mean -2SD) and four exhibited catch-up growth. Thus, steroid withdrawal can be safe and improves growth in pediatric renal transplant recipients if undertaken with careful clinical follow-up, including protocol biopsies.

Biopsy↗
PubMed1999

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

Acetylglucosamine↗
PubMed1999

Chemotherapy with hybrid liposomes for melanomatosis.

The inhibitory effects of the hybrid liposomes on the growth of B-16 melanoma cells in vitro and in vivo were examined. The 50% inhibitory concentration of the hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylenedodecyl ether (C12(EO)10) was one-twelfth of that of DMPC liposomes. It was noteworthy that for the first time significantly prolonged survival was obtained using a mouce model of carcinoma after the administration of the hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n=10 or 23) without antitumor drugs.

Animals↗
PubMed1999

Photodynamic therapy of atherosclerosis using YAG-OPO laser and Porfimer sodium, and comparison with using argon-dye laser.

We performed photodynamic therapy (PDT) using the Yttrium Aluminium Garnet-Optical Parametric Oscillated (YAG-OPO) laser in cases of atherosclerosis, and examined its efficacy in vivo. We also performed PDT using an Argon-dye (Ar-dye) laser with the same output, and compared the efficacies. Following balloon denudation injury of the thoracoabdominal aorta, rabbits were raised on a cholesterol diet for 16 weeks, producing atheroma in that region. At 24 h following the administration of Photofrin 5 mg/kg, PDT was performed, and animals were sacrificed at 1 day, 1 week, and 2 weeks following the procedure to examine its efficacy. This was compared with the efficacy of PDT using the Ar-dye laser. Following PDT using a YAG-OPO laser, an increase in the vessel lumen was seen due to reduction of the hypertrophic intima and media, without the appearance of inflammatory cells. This result was seen more strongly in PDT using the pulse wave YAG-OPO laser than with the continuous wave Ar-dye laser, affecting not just the intima but also the media. These data demonstrated that PDT can effectively regress atherosclerotic lesions.

Animals↗
PubMed1999

Fluorescence spectrum analysis of atherosclerotic plaque using doxycycline.

Using doxycycline (DOXY), fluorescence spectrum analysis was performed on arteriosclerotic lesions, and the efficacy of this method was examined in basic and clinical studies. In the basic study, DOXY 50 mg was administered intravenously to arteriosclerotic rabbits, and the thoracoabdominal aorta removed. Fluorescence spectral analysis was performed on each specimen, and the fluorescence spectral pattern, peak intensity and degree of intimal hypertrophy were studied. In the clinical study, DOXY 200 mg was administered intravenously to 6 human subjects with stable angina and coronary arterial stenosis of greater than 90%, and coronary angiography, coronary angioscopy and fluorescence spectral analysis were performed. DOXY accumulation in the arteriosclerotic intima of rabbit aortae was confirmed. The fluorescence spectrum was monomodal, peaking at around 532 nm. In the noncalcification group, significant correlation was observed between peak intensity and arteriosclerotic intimal thickness. Using DOXY as a fluorescent marker, it was possible to assess the level of arteriosclerotic intimal hypertrophy. Clinically, it was possible to obtain the DOXY spectrum of the coronary arteries.

Animals↗
PubMed1999

A study to determine if basic fibroblast growth factor (bFGF) reduces myocardial infarct size in acute coronary arterial occlusion.

We investigated the angiogenic and myocardial salvage effects of bFGF. Twelve beagles with ligated left anterior descending coronary arteries were divided into two groups: a FGF group administered bFGF intravenously, and a Control group, after CAG immediately post-ligation. One week post-ligation, CAG was repeated. The heart was sliced along the short axis. For each section, the fluorescein Na staining deficit area (DA) and ratio of DA to total area (DAR), TTC staining of the infarct area (IA) and ratio of IA to total area (IAR), and Masson trichrome staining of the fibrosed area (MA) and ratio of MA to total area (MAR), were calculated. The increase in the number of collateral vessels, seen on CAG from post-ligation to 1 week later, was significantly greater in the FGF group. No significant differences in IAR or MAR were seen between the groups. However, DAR and DA/IA were significantly less in the FGF group. In conclusion, bFGF had no effect on infarct size, but stimulated the growth of collateral vessels and improved coronary blood flow in IA.

Animals↗
PubMed1999

Effects of sex hormones on production of interleukin-1 by human peripheral monocytes.

BACKGROUND: Interleukin-1 (IL-1) is a potent mediator of inflammation and is known to induce bone resorption. We studied the effects of sex hormones on the function of human monocytes and demonstrated that prostaglandin E2 (PGE2) production was enhanced by progesterone and estradiol. As PGE2 has been shown to suppress the production of IL-1 by monocytes, it was speculated that sex hormones also modify the production of IL-1 by regulating PGE2 production. Thus, the effects of sex hormones on the production of IL-1 from human peripheral monocytes and the influence of PGE2 were investigated. METHODS: Mononuclear leukocytes were obtained from 22 healthy adults. Progesterone, 17-beta estradiol (estradiol), and testosterone were used as representative sex hormones. Monocytes were incubated at 37 degrees C in air with 5% CO2 for 24 hours in RPMI 1640 medium with sex hormones at the designated concentrations. LPS (Salmonella typhimurium) was used to stimulate the monocytes at a concentration of 10 microg/ml. The concentrations of IL-1alpha and IL-1beta in the medium were determined by enzyme-linked immunosorbent assay kits. The concentration of PGE2 was determined using a direct radio-immunoassay kit. Indomethacin was used to inhibit the synthesis of PGE2 and eliminate its effect on the production of IL-1. RESULTS: Estradiol at concentrations of 0.04 ng/ml or more significantly reduced both IL-1alpha and IL- 1beta production. Progesterone also reduced IL-1alpha and IL-1beta production significantly at concentrations of 0.1 ng/ml or more and 0.02 ng/ml or more, respectively. The reductions in IL- 1alpha and IL-1beta production by sex hormones were not affected by addition of indomethacin. CONCLUSIONS: Estradiol and progesterone inhibited the production of IL-1 from human peripheral monocytes. The inhibition was not the result of enhanced production of PGE2. Under conditions in which sex hormone levels are low, monocytes produce IL- more readily in response to stimulation by LPS than high levels of such hormones. Low concentrations of sex hormones may be considered as one of the risk factors for periodontitis.

Adult↗
PubMed1999

Characterization of urinary metabolites of a new benzofuroquinoline derivative, 3,9-bis(N,N-dimethylcarbamoyloxy)-5 H-benzofuro[3,2-c]-quinoline-6-one (KCA-098), in dogs.

The metabolism of 3,9-bis(N,N-dimethylcarbamoyloxy)-5 H-benzofuro[3,2-c]-quinoline-6-one (KCA-098), a new inhibitor of bone resorption and stimulator of bone formation, was examined after oral administration to dogs. Nine metabolites and the unchanged KCA-098 were isolated by extraction and HPLC from dog urine. The structures of these metabolites were characterized by LC/MS or LC/MS/MS, and/or were confirmed by comparison with corresponding authentic standards. The presumed main metabolic pathways were hydrolysis, hydroxylation, and N-demethylation of the N,N-dimethyl-carbamate ester group.

Animals↗
PubMed1999

[Relation between temporomandibular joint disorder and subjective symptoms of fatigue].

An investigation was performed for the purpose of guaging the relation between temporomandibular joint disorder on the one hand and occupational category and subjective symptoms of fatigue on the other. Subjects were 2,414 workers (1,598 male, 816 female) in small and medium size enterprises at 67 workplaces in Hiroshima Prefecture. A comparison of cases with and without temporomandibular joint disorder showed that there were more complaints of fatigue among those with the disorder. Given these indications that temporomandibular joint disorder was related to occupation, it is possible that temporomandibular joint disease may be a work-related disease.

Adult↗
PubMed1999

A case of ABO-incompatible renal transplant patient with non-insulin-dependent diabetes mellitus; long-standing observation of serial glomerular change by protocol biopsy.

A 41-yr-old patient with non-insulin-dependent diabetes mellitus (NIDDM), before and after ABO-incompatible renal transplant, is reviewed using serial protocol biopsy. Although she recovered from delayed hyperacute rejection (DHAR) immediately post-transplantation, her graft function deteriorated gradually. A mild acute transplant glomerulitis, noted at the 155th day post-transplantation, progressed to pronounced chronic transplant glomerulopathy over 5 yr. In the specimen of the last biopsy, at 5 yr post-transplantation, glomeruli demonstrated an exudative hyaline lesion, which was characteristic of diabetic nephropathy in addition to chronic transplant glomerulopathy. Therefore, we made a diagnosis of this glomerular lesion as chronic transplant glomerulopathy complicated by diabetic glomerulopathy. Considering the result of this case, the protocol biopsy is a useful procedure to diagnose an accurate cause of graft dysfunction in individual cases. It is concluded that the protocol biopsy is apparently useful for the detection of various pathological processes occurring in allograft and may contribute to a strategy for improvement of graft survival.

ABO Blood-Group System↗
PubMed1999

Er:YAG and alexandrite laser radiation propagation in root canal and its effect on bacteria.

OBJECTIVE: The objective of this study was to compare the bactericidal effect of the Er:YAG (wavelength 2.94 microm) and the Alexandrite (wavelength 0.75 microm) laser radiation. The spreading laser energy in the surrounding hard dental tissues round the root canal was evaluated and the bactericidal effect of both these different laser wavelengths was analyzed. SUMMARY BACKGROUND DATA: The use of a laser to clean and shape the root canal space is the latest method used for cleaning of root canals. The interest in laser endodontics was concentrated on the possibility to extirpate the contents of the root canal, to sterilize and to "melt" the walls of the root canals. The previous reports were performed with CO2, excimer, argon, Nd:YAG, Ho:YAG, and Er:YAG lasers. METHODS: Er:YAG laser system (2.94 microm, energy 100 mJ or 300 mJ, repetition rate 1 Hz, 30 pulses) and alexandrite laser system (0.75 microm, energy 250 mJ, repetition rate 1 Hz, 30 pulses) were prepared and three experimental arrangements were used during the measurements. First the energy transport through the tooth tissue was observed (frontal and side experimental setups) and then, the bactericidal effect was evaluated. RESULTS: It was demonstrated that due to the absorption in the hydroxyapatite and water content in the dentin, the Er:YAG laser radiation is fully absorbed in the root canal wall. This direct influence of the radiation could be expected only close to the sapphire tip. It was found that the tissue, which was not directly affected by the laser radiation, cannot be disinfected by the subablative effect of Er:YAG laser radiation. In the second part of the experiment the real bactericidal effect of Er:YAG ablative energy (300 mJ) could be observed. It was also shown that the alexandrite laser radiation with a wavelength of 0.75 microm spreads through the canal system space and leaks into the surrounding tooth tissues. Both lasers have bactericidal effect. CONCLUSIONS: The pulsed Er:YAG and alexandrite lasers can be efficiently used for killing dental bacteria but the spreading of their radiation in the tooth tissues are different.

Dental Pulp Cavity↗
PubMed1998

Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B.

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

Acetates↗