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Biomedical subjects

N Hirayama

Publications and source records attributed to N Hirayama.

At least 37 records · Page 2Linked to original sources
PubMed1999

Alteration of substrate affinity of Streptomyces cholesterol oxidase for application to the rate assay of cholesterol in serum.

The Streptomyces cholesterol oxidase (ChoA) can not be adopted for the rate assay, because the Km value of the enzyme for cholesterol is very small. The choA gene was subjected to random mutagenesis in vivo, and a mutant ChoA (designated E-ChoA) that showed altered substrate affinity was obtained by screening. The Km value of E-ChoA was approximately ten times larger than that of the wild type. Unexpectedly, the thermal stability was also improved. The amino acid substitutions of E-ChoA were identified to be the valine to glutamate at position 145, which has been previously identified as one of the thermostable mutations, and the glycine to serine at position 405. The mutational effects on the structure of E-ChoA are discussed on the basis of a three-dimensional model. E-ChoA has been successfully applied to the rate assay of cholesterol in serum.

Cholesterol↗
PubMed1999

Secretion and clearance of the mature form of adrenomedullin in humans.

In the biosynthesis of adrenomedullin (AM), glycine-extended AM, an intermediate form (iAM) processed from proAM is converted to AM[1-52]-NH2, the bioactive mature form of AM (mAM), by enzymatic amidation. We earlier showed that both molecular forms of AM circulate in human plasma. In the present study, to investigate the secretion and clearance sites of mAM and iAM in humans, we examined the plasma mAM and iAM concentrations in the femoral artery and vein (FA and FV), the aortic root and coronary sinus (AO and CS), and the pulmonary artery and capillary (PA and PC) of patients with ischemic heart disease. Plasma mAM in FV was significantly (p<0.001) higher than in FA. There also was a significant (p<0.001) step-up in the plasma mAM of the CS as compared to the AO. In contrast, plasma mAM was significantly (p<0.001) reduced in the PC as compared to the PA. However, such differences were not observed in plasma iAM levels. These findings suggest that in humans the vasculature of the lower extremities and the heart produce and secrete mAM and that the lung is a clearance site of circulating mAM.

Adrenomedullin↗
PubMed1999

Epitopes and nuclear localization analyses of canine distemper virus nucleocapsid protein by expression of its deletion mutants.

A series of nucleocapsid protein (NP)-deleted genes of the Onderstepoort strain was constructed in order to locate antigenic regions of the NP of canine distemper virus. The expression of proteins from 5'-deleted NP genes was examined in COS-7 cells by indirect immunofluorescence assay using three monoclonal antibodies (MAbs), c-5, f-5 and h-6, and a rabbit serum against NP. These MAbs reacted with two regions of NP. Amino acid residues from 1 to 80, and 337-358, were necessary and sufficient for formation of the epitopes identified by MAbs f-5 and h-6, and c-5, respectively. The proteins translated from intact or 3'-deleted genes were found to be localized in the nuclei of COS-7 cells, whereas the proteins from the 5'-deleted genes were mainly detected in the cytoplasm. These results suggested that 80 amino acid residues at the N-terminus are required for transportation of NP into the nucleus.

Animals↗
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

Use of super-water-repellent fractal surfaces in the crystallization of macromolecules.

The effect of fractal surfaces on protein crystallization has been investigated. A super-water-repellent fractal surface consisting of alkylketene dimers was formed on microscope cover slips. Crystallization experiments with hen egg-white lysozyme using the sitting-drop vapour-diffusion method have been undertaken on the fractal and conventionally siliconized surfaces. The size of the crystals obtained on the fractal surface is significantly larger than on the conventionally siliconized surface. The number of crystals on the fractal surface is markedly smaller than on the conventionally siliconized surface. The results clearly indicate that the fractal surface is very effective and promising in the crystallization of macromolecules using the sitting-drop vapour-diffusion method.

Animals↗
PubMed1999

Molecular forms of circulating adrenomedullin in patients with congestive heart failure.

In the biosynthesis of adrenomedullin (AM), an intermediate form, AM(1-52)-glycine-COOH (iAM), is cleaved from proAM and subsequently processed to a biologically active mature form, AM(1-52)-NH2 (mAM), by enzymatic amidation. We recently reported that immunoreactive AM in human plasma consists of mAM and iAM. To clarify the pathophysiological roles of mAM and iAM in heart failure, we established an assay method to specifically detect mAM, and we determined the plasma concentrations of mAM and iAM in 68 patients with congestive heart failure (CHF). The plasma mAM concentrations of the CHF patients classified as being class I or II of New York Heart Association (NYHA) functional classification were significantly greater than those of the 28 healthy controls, and a further increase was noted in the class III or IV patients. Similar increases in plasma iAM were also observed in these patients compared with controls. The increased plasma mAM and iAM in 12 patients with exacerbated CHF were significantly reduced by treatment of their CHF for 7 days. In addition, the plasma concentrations of both mAM and iAM were significantly correlated with pulmonary capillary wedge pressure, pulmonary artery pressure, right atrial pressure, cardiothoracic ratio, heart rate, and the plasma concentrations of atrial and brain natriuretic peptides in the CHF patients. Thus the plasma concentrations of both mAM and iAM were increased progressively in proportion to the severity of CHF. These results suggest that, though the role of iAM remains to be clarified, mAM acts against the further deterioration of heart failure in patients with CHF.

Adrenomedullin↗
PubMed1999

Increased plasma levels of mature form of adrenomedullin in patients with chronic renal failure.

BACKGROUND: Adrenomedullin (AM), a novel vasodilator peptide, is produced by C-terminal amidation reaction of AM-glycine. AM-glycine, an intermediate form of AM (iAM), is processed from pro AM. AM circulating in the human blood stream was found to consist of an amidated mature form (mAM) and iAM. Biological activity is exerted only by mAM. METHODS: To investigate the pathophysiological role of mAM in renal disease, we measured plasma concentrations of mAM as well as total AM (tAM), representing both mAM and iAM, in patients with various renal diseases. In addition, plasma ANP level was measured in all patients. RESULTS: The concentrations of plasma mAM in renal failure with dialysis (2.1 +/- 0.2 fmol/ml, mean +/- SEM) and without dialysis (1.2 +/- 0.2) were significantly (p < 0.05) higher than those in control group (0.5 +/- 0.1). However, the plasma ANP level was increased only in renal failure patients with dialysis. Plasma mAM levels were significantly correlated positively with serum creatinine levels and negatively with hematocrit. No significant difference was noted in the ratio of mAM/tAM between renal failure patients and healthy subjects. CONCLUSION: These results suggest that plasma mAM is increased in renal failure in relation to deterioration of renal function, while the amidation process of AM seems to be unaffected in patients with renal failure.

Adrenomedullin↗
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↗
PubMed1998

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase.

Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.

Amino Acids↗
PubMed1998

Effect of non-ionic detergents on apparent enzyme mechanism: V121A mutant of Streptomyces cholesterol oxidase endowed with enhanced sensitivity towards detergents.

One of the mutants of Streptomyces cholesterol oxidase with the Val121Ala mutation (V121A) was kinetically analysed. Although the reaction rate-substrate concentration curve of wild type follows a simple Michaelis-Menten equation, that of V121A is sigmoidal. The cooperativity was apparent and caused by non-ionic detergents that were used as a solvent of cholesterol. The concentration dependence of V121A on detergents was more significant than that of wild type, although the reaction rates of both enzymes decrease as the concentrations of detergents increase. Further experiments suggested that less hydrophobic interactions between V121A and detergents should be responsible for the apparent cooperativity. Since Val121 is in a hydrophobic loop located near the active site, the mutational effect is structurally discussed.

Binding Sites↗
PubMed1998

Incidence of urogenital cancers in Gunma Prefecture, Japan: a 10-year summary.

BACKGROUND: Although the incidence of urogenital cancers in Japan is lower than that of other cancers, it is increasing steadily. Thus, an epidemiologic study was necessary to determine the measures that would decrease the mortality rate associated with these cancers. METHODS: The subjects were 4759 patients with urogenital cancer who were living in Gunma Prefecture and who were newly diagnosed between 1985 and 1994. The data were analyzed by year and by patients' ages. The incidence rates of each disease were expressed as the number of cases per 100,000/year, and age-adjusted rates were adjusted to the world population. RESULTS: The number of males and females afflicted by urogenital cancers increased over the 10-year period. The increase in age-adjusted incidence rates was sharpest for prostate, renal cell, and testicular cancers among males, and for renal cell, renal pelvic and ureter cancers among females. When age-specific rates were plotted against age on double logarithmic scales, the cancers were classified as type 1 (linear), type 2 (linear until a certain age, then flattening out or decreasing), or type 3 (irregular) based on the slope of the line. The magnitude of increase in the age-specific incidence rates of type 1 cancers with age was on the order of the 12th power for prostate cancer and the 5th power for bladder cancer. When the 10 years were divided into 2 periods (earlier and later), the age-specific incidence rates of prostate and renal cell cancers increased in all age groups, whereas the age-specific incidence rates of cancers that increased less sharply remained stable or even declined in some age groups. CONCLUSION: These epidemiologic data should be useful in reducing the mortality rates associated with these cancers.

Adult↗
PubMed1997

Structure of KW-2228, a tailored human granulocyte colony-stimulating factor with enhanced biological activity and stability.

KW-2228 is a tailored human granulocyte colony-stimulating factor (hG-CSF) which has more potent granulopoietic activity and is more stable than wild-type hG-CSF. Analysis of the 2.3 A resolution crystal structure of KW-2228 unambiguously revealed a four-alpha-helix bundle motif with up-up-down-down connectivity. The structures of long overhand loops connecting the helices and the N-terminus have been definitively determined. The present analysis has clearly revealed that substituted residues play important roles in fastening a long overhand loop to the N- and C-termini to fix the conformation. This conformation should be responsible for a substantial enhancement of the biological activity and stability.

Crystallography, X-Ray↗
PubMed1997

Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation.

Random mutagenesis was used to enhance the thermal stability of Streptomyces cholesterol oxidase. Four thermostable mutants were isolated and the following amino acid substitutions were identified: Ser103 to Thr (mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H) and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were purified and characterized. The properties of mutants S103T, V121A and R135H were similar to those of the wild type but they showed improved thermal stability. When the V145E mutation was introduced, the thermal stability of the enzyme was markedly increased and the optimum pH was desirably changed to encompass a broad range from acid to alkali. Analysis of multiple mutants constructed by site-directed mutagenesis showed that all the mutations except that of R135H had an additive influence on the other mutations. These mutational effects are discussed in terms of a three-dimensional structural model of the enzyme constructed on the basis of homology modelling.

Cholesterol↗
PubMed1997

Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs.

We isolated three strains of canine distemper virus (CDV)--the Ueno, Hamamatsu, and Yanaka strains--from dogs in Japan and analysed the molecular properties of their haemagglutinin (H) proteins. Immunoprecipitation of all three strains with a monoclonal antibody revealed H proteins with molecular masses of 84 kDa, which differs from the molecular mass (78 kDa) of the H protein of the Onderstepoort vaccine strain. However, after tunicamycin treatment immunoprecipitation identified H proteins of identical molecular mass (68 kDa) for all three field isolates and the vaccine strain. Sequence analysis showed nine potential sites for asparagine-linked glycosylation in the H proteins of the new isolates, in contrast to four in the H protein of the Onderstepoort strain. Thus, variation in glycosylation of the H proteins of the isolates and the vaccine strain may cause differences in antigenicity of the viruses. Sequences of the H genes showed that the new Japanese isolates have 99% identity with each other, 95% with other European and American isolates (from seals, a German dog, a ferret and large felids) and 90% with the vaccine strain. Phylogenetically, the new Japanese isolates form one cluster which is separate from recent European or American isolates, all of which are distinct from vaccine strains.

Amino Acid Sequence↗
PubMed1996

Structural studies of mitomycins. VIII. Mitomycin D hydrate, C15H18N4O5.1.5H2O.

The title compound, [1aS]-6-amino-1, 1a,2,4,7,8,8a,8b-octahydro-8a-hydroxy-1,5-dimethyl-4,7-dioxoazirino++ +[2',-3': 3,4]pyrrolo[1,2-a]indol-7-ylmethyl, is a mitomycin derivative, mitomycins being antitumor antibiotics. The O atoms of the quinone ring deviate significantly from the least-squares plane through the quinone ring.

Antibiotics, Antineoplastic↗