Biomedical subjects

M Otagiri

Publications and source records attributed to M Otagiri.

At least 19 recordsLinked to original sources

PubMed2005

Renal clearance of glycolaldehyde- and methylglyoxal-modified proteins in mice is mediated by mesangial cells through a class A scavenger receptor (SR-A).

AIMS/HYPOTHESIS: Glomerular mesangial expansion is a characteristic feature of diabetic nephropathy, and the accumulation of AGE in the mesangial lesion has been implicated as one of its potential causes. However, the route for the AGE accumulation in mesangial lesions in diabetic patients is poorly established. METHODS: Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were prepared as model AGE proteins, and their in vivo plasma clearance was examined in mice, and renal uptake by in vitro studies with isolated renal mesangial cells. RESULTS: Both (111)In-GA-BSA and (111)In-MG-BSA were rapidly cleared from the circulation mainly by both the liver and kidney. Immunohistochemical studies with an anti-GA-BSA antibody demonstrated that intravenously injected GA-BSA accumulated in mesangial cells, suggesting that such cells play an important role in the renal clearance of circulating AGE proteins. Binding experiments at 4 degrees C using mesangial cells isolated from mice showed that (125)I-GA-BSA and (125)I-MG-BSA exhibited specific and saturable binding. Upon incubation at 37 degrees C, (125)I-GA-BSA and (125)I-MG-BSA underwent endocytic degradation by these cells. The binding of the ligands to these cells was inhibited by several ligands for scavenger receptors. The endocytic degradation of GA-BSA by mesangial cells from class A scavenger receptor (SR-A) knock-out mice was reduced by 80% when compared with that of wild-type cells. The glomerular accumulation of GA-BSA after its intravenous administration was attenuated in SR-A knock-out mice, as evidenced by immunohistochemical observations. CONCLUSIONS/INTERPRETATION: These results raise the possibility that circulating AGE-modified proteins are subjected to renal clearance by mesangial cells, mainly via SR-A. This pathway may contribute to the pathogenesis of AGE-induced diabetic nephropathy.

Acetaldehyde↗

PubMed2002

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat.

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.

Alcohol Oxidoreductases↗

PubMed2001

Up-regulation by clarithromycin of alpha(1)-acid glycoprotein expression in liver and primary cultured hepatocytes.

alpha(1)-Acid glycoprotein (AGP) is the major transport protein for cationic drugs, endogenous ligands, and some anionic drugs in plasma. Hepatic synthesis and secretion of AGP are altered during acute inflammation as well as by a number of drugs. This alteration could influence the binding of drugs and its biological function. Macrolide antibiotics are widely used in the treatment of a variety of infectious diseases. The effects of macrolide antibiotics have been studied with respect to rat AGP expression in vivo. After the individual administration of six macrolides to rats, with the exception of oleandomycin, five increased AGP levels in serum. Of these five, clarithromycin (CAM) was the most potent inducer of AGP, which reached a maximum level between 3 to 7 days after administration. CAM increased the steady-state level of AGP mRNA in liver as well as protein level in serum in a dose-dependent manner. In addition, CAM increased AGP mRNA levels in primary cultured hepatocytes. In the luciferase promoter assay, CAM potentiated dexamethasone-increased promoter activity of the AGP gene, which contained the glucocorticoid response element, in cultured rat hepatocytes, although CAM itself had no effect on its activity. The effect of CAM and dexamethasone was diminished by glucocorticoid response element deletion or mutation or by adding the antiglucocorticoid, RU486. Further, in the mouse mammary tumor virus (MMTV) promoter containing functional glucocorticoid response element, CAM potentiated dexamethasone-increased promoter activity. In the adrenalectomized rats, CAM did not increase AGP levels in serum. These findings suggest that CAM may cause transcriptional induction of AGP, at least in part, via a glucocorticoid-mediated mechanism.

5' Untranslated Regions↗

PubMed2001

Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought.

Amino Acid Substitution↗

PubMed2001

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma.

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCI were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)-acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.

Animals↗

PubMed2001

FK506 (tacrolimus) increases rat alpha1-acid glycoprotein expression in liver and primary cultured hepatocytes.

FK506 (tacrolimus) (10 mg/kg, s.c., 5 days) increased rat alpha1-acid glycoprotein (AGP) in serum and AGP mRNA in liver. FK506 potentiated the dexamethasone-increased AGP expression in primary cultured hepatocytes. In the luciferase promoter assay, FK506 potentiated the dexamethasone-increased promoter activity of the AGP gene in cultured rat hepatocytes, although FK506 alone had no effect on its activity. The combined effect of FK506 and dexamethasone was diminished by glucocorticoid responsive element (GRE) deletion and mutation or by an anti-glucocorticoid. These results indicated that FK506 causes the transcriptional induction of AGP, at least in part, via a glucocorticoid-mediated mechanism.

Animals↗

PubMed2001

Crystallization and preliminary X-ray studies of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae IAM1063.

Meso-2,3-butanediol dehydrogenase (meso-BDH) has been crystallized and preliminary X-ray crystallographic characterization of meso-BDH crystals has been performed. Single crystals of meso-BDH were prepared in two forms by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Form I crystals belong to space group C2, with unit-cell parameters a = 215.5, b = 79.4, c = 134.8 A, beta = 98.22 degrees, and form II crystals belong to space group P2(1), with unit-cell parameters a = 69.16, b = 109.78, c = 127.28 A, beta = 102.29 degrees. The crystals diffracted to 2.0 and 1.7 A resolutions, respectively, using synchrotron radiation.

Alcohol Oxidoreductases↗

PubMed2001

Flunitrazepam, a 7-nitro-1,4-benzodiazepine that is unable to bind to the indole-benzodiazepine site of human serum albumin.

Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.

Anti-Anxiety Agents↗

PubMed2001

Determination of reduced, protein-unbound, and total concentrations of N-acetyl-L-cysteine and L-cysteine in rat plasma by postcolumn ligand substitution high-performance liquid chromatography.

A high-performance liquid chromatographic assay was developed for the quantitative determination of the sulfur-containing amino acids N-acetyl-L-cysteine (NAC) and L-cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuously mixed with an iodoplatinate-containing solution. The substitution of sulfur of the thiol compound with iodide was quantitatively determined by measuring changes in the absorption at 500 nm. The low-molecular-weight disulfides and mixed disulfide conjugates of thiols with proteins were entirely reduced to the original reduced compounds by dithiothreitol. By reducing these two types of disulfides separately during sample pretreatment, the reduced, protein-unbound, and total thiol concentrations could also be determined. Validation testing was performed, and no problems were encountered. The limit of detection was approximately 20 pmol of thiol on the column. The present method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAC, focusing on disulfide formation. The binding of NAC to protein through mixed disulfide formation proceeds in a time-dependent and reversible manner. Moreover, this "stable" covalent binding might limit total drug elimination, while the unbound NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, including disulfide conjugates derived from them.

Acetylcysteine↗

PubMed2001

Hormonal regulation of male-specific 20beta-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in kidney microsomes of rats.

Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which are C(21)-steroids with a ketone group at the 20-position, potently inhibited the activity of enzyme acetohexamide reductase (AHR) responsible for the reductive metabolism of acetohexamide in kidney microsomes of male rats. Furthermore, progesterone was a competitive inhibitor of AHR. In the case of progesterone usage as the substrate, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) activity was much higher than 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity in kidney microsomes of male rats. These results indicate that AHR present in kidney microsomes of male rats, functions as 20beta-HSD with carbonyl reductase-like activity. In male rats, both testectomy and hypophysectomy decreased the renal microsomal 20beta-HSD activity, but the decreased enzyme activities were increased by the treatment with testosterone propionate (TP). We propose the possibility that TP treatment regulates the renal microsomal 20beta-HSD activity by acting directly on the kidney of male rats. This is supported from the fact that when TP was given to ovariectomized and hypophysectomized female rats, the male-specific 20beta-HSD activity was detected in their kidney microsomes.

Alcohol Oxidoreductases↗

PubMed2001

Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin.

PURPOSE: The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. METHODS: Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. RESULTS: The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: "competitive-like" strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly "independent". CONCLUSIONS: The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.

Anticoagulants↗

PubMed2001

Effect of oxidative stress on the structure and function of human serum albumin.

PURPOSE: Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esterase-like properties were studied. METHODS: Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p-nitrophenyl acetate as a substrate. RESULTS: Far-UV and near-UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO-HSA and CT-HSA but not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross-linking and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO-HSA and CT-HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT-HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level. CONCLUSIONS: Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase-like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

Amino Acids↗

PubMed2001

In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time.

PURPOSE: Recombinant human serum albumin (rHSA), secreted by a Pichia pastoris expression system, was purified by a fast and efficient method, the essential feature of which is strong but reversible binding of the protein to Blue Sepharose. The structural characteristics, stability, and ligand-binding properties of the resulting protein were examined, and pre-clinical studies were performed. METHODS: Protein structure was investigated by amino acid sequencing, sodium polyacrylamide gel electrophoresis, CD spectroscopy and chromatography. Stability was examined by denaturation by guanidine hydrochloride and by calorimetry, and ligand binding was studied by ultrafiltration. Rat experiments were performed with 125I-labeled albumin. RESULTS: Far-ultraviolet and near-ultraviolet CD spectra of rHSA were identical to those of human serum albumin isolated from serum (HSA). Mercaptalbumin and non-mercaptalbumin were separated by high-performance liquid chromatography using an N-methylpyridinium polymer-based column. 60% of rHSA existed as mercaptalbumin, a content that is higher than that of a commercial preparation of HSA. Fatty acids, N-acetyl-L-tryptophan and pasteurization had similar effects on the conformational stability of rHSA and HSA. Stereoselective ligand-binding properties (warfarin, phenprocoumon, pranoprofen and ibuprofen) of rHSA were the same as those of HSA. The effect of the neutral to base transition on warfarin (site I-ligand) and dansylsarcosine (site II-ligand) binding to rHSA was also similar to HSA. In vivo studies showed comparable half-lives, excretion and tissue distributions of the two albumin preparations. CONCLUSION: The present yeast expression system and purification procedure result in rHSA with structural and functional properties very similar to those of HSA.

Animals↗

PubMed2001

Protective effects of suprofen and its methyl ester against inactivation of rabbit kidney carbonyl reductase by phenylglyoxal.

Suprofen (SP) was little reduced by rabbit kidney carbonyl reductase, whereas its methyl ester (SPM) was an efficient substrate of the enzyme. To account for the differential catalytic activities for SP and SPM, the protective effects of these compounds against the inactivation of the enzyme by phenylglyoxal (PGO) were compared. Since the carboxyl group of SP is negatively charged and one essential arginine residue is known to be located in the NADPH-binding site of the enzyme, the protection of SP against the inactivation of the enzyme by PGO is expected to be more effective than that of SPM lacking a carboxyl group. However, the protective effects of SP and SPM were very similar. These results suggest that in spite of evidence for the binding of SP to the coenzyme-binding site, the carboxyl group of SP fails to interact with one essential arginine residue located in the site.

Alcohol Oxidoreductases↗

PubMed2001

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms.

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.

Alcohol Oxidoreductases↗

PubMed2001

Stability of a cisplatin-chondroitin sulfate A complex in plasma and kidney in terms of protein binding.

To assess the stability of a cisplatin (CDDP) complex prepared with chondroitin sulfate A (CSA) relative to protein binding in the circulation and kidney, a trichloroacetic acid (TCA) precipitation method was developed to measure the protein-unbound species of CDDP and the CDDP-CSA complex in plasma and kidney homogenates. The total and unbound drug concentrations were determined up to 3 h following a 2 mg/kg bolus injection of CDDP or CDDP-CSA complex to rats. The stability against plasma binding was evaluated by a determination of the area under concentration-time curve from time 0 to infinite time (AUC(0-infinity)); the ratio of unbound drug AUC(0-infinity) to total drug AUC(0-infinity) was employed to estimate the availability of the unbound drug in the circulation. The results showed that a competitive reaction to platinum existed between plasma protein and the CDDP-CSA complex, but the complex accounted for more than 60% of the protein-unbound species for a dosage, compared to 30% obtained by an administration of uncomplexed CDDP. The tissue binding kinetics in kidney for CDDP and the CDDP-CSA complex was investigated by the use of homogenates. The binding rate constants of CDDP and CDDP-CSA in a kidney homogenate were 0.0040 min(-1) and 0.0014 min(-1), respectively. The results indicate that the CDDP-CSA complex could effectively retard the binding of CDDP to protein in the kidney. These data provide evidence that endogenous protein is able to compete for platinum from the CDDP-CSA complex, but the complex effectively retarded the protein binding reaction with CDDP in plasma and kidney as compared to native CDDP, which has the potential for reducing the accumulation of CDDP in plasma and kidney.

Animals↗

PubMed2001

[Strain-, sex- and species-related differences of acetohexamide reductase and 20 beta-hydroxysteroid dehydrogenase activities in liver microsomes of experimental animals].

We examined physiological and genetic factors affecting acetohexamide reductase (AHR) and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) activities in liver microsomes of experimental animals. Pronounced strain-related differences were found in both activities of AHR and 20 beta-HSD present in liver microsomes of male rats. Among rat strains tested in this study, even though a Wistar-Imamichi (WIM) rat strain was taken to lack AHR activity, it exhibited a significant 20 beta-HSD activity. These findings appeared to be in conflict with our conclusion reported so far, which AHR and 20 beta-HSD present in liver microsomes of male rats are identical enzymes. Thus the reason for this discrepancy was discussed. Furthermore, AHR and 20 beta-HSD activities were little or not observed in liver microsomes of female rats or male experimental animals other than the rat, indicating the existence of sex- and species-related differences in these two enzyme activities.

20-Hydroxysteroid Dehydrogenases↗

PubMed2001

Purification and characterization of L-2,3-butanediol dehydrogenase of Brevibacterium saccharolyticum C-1012 expressed in Escherichia coli.

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.

Alcohol Oxidoreductases↗