Biomedical subjects

M Takano

Publications and source records attributed to M Takano.

At least 127 records · Page 7Linked to original sources

PubMed1998

Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells.

The effect of cytochrome P-450 3A (CYP3A) substrates (erythromycin, midazolam) and an inhibitor (ketoconazole) on P-glycoprotein-mediated transport was studied in Caco-2, the human colon adenocarcinoma cell line expressing various functions of differentiated intestinal epithelial cells. The involvement of P-glycoprotein in the transport of these drugs was also examined. The basal-to-apical transport of rhodamine 123, a P-glycoprotein substrate, was inhibited by erythromycin, midazolam and ketoconazole, as well as by P-glycoprotein inhibitors such as verapamil. The apical-to-basal transport of rhodamine 123 was increased by these drugs. The transepithelial transport of erythromycin and midazolam, but not of ketoconazole, was much greater from the basal to apical side than from the apical to basal side. The inhibitory effect of verapamil was observed on the basal to apical transport of erythromycin, but not on midazolam and ketoconazole transport. In conclusion, erythromycin, midazolam and ketoconazole could interact with P-glycoprotein-mediated transport, and P-glycoprotein could be, at least in part, involved in the transport of erythromycin, but not of midazolam and ketoconazole, in the intestinal epithelia.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1998

Kininogen expression by rat vascular smooth muscle cells: stimulation by lipopolysaccharide and angiotensin II.

To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.

Angiotensin II↗

PubMed1998

Prostaglandin E2 protects against liver injury after Escherichia coli infection but hampers the resolution of the infection in mice.

cAMP-increasing agents such as prostaglandin E2 (PGE2) are known to protect against LPS-induced liver injury by downregulating the production of inflammatory cytokines such as TNF-alpha. However, the effects of such reagents on host defense against bacterial infection remain unknown. We show here that in vivo administration of PGE2 significantly protected mice against liver injury after Escherichia coli infection but hampered the resolution of the infection. PGE2 significantly suppressed circulating TNF-alpha and IL-12 levels but increased the IL-10 production after E. coli challenge. PGE2 inhibited the emergence of gammadelta T cells in the peritoneal cavity, which are important for host defense against E. coli, and deteriorated bacterial exclusion in the peritoneal cavity after E. coli challenge. These results suggested that PGE2 affects host defense mechanisms against E. coli infection through modulation of cytokine production and gammadelta T cell accumulation.

Animals↗

PubMed1998

Ambient but not incremental oxidant generation effects intercellular adhesion molecule 1 induction by tumour necrosis factor alpha in endothelium.

Proinflammatory cytokines upregulate endothelial adhesion molecule expression, thereby initiating the microvascular inflammatory response. We re-evaluated the reported role of reactive oxygen metabolites (ROMs) in signalling upregulation of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells by tumour necrosis factor alpha (TNF-alpha) in vitro. TNF-alpha upregulation of endothelial-cell ICAM-1 expression was inhibited by the cell-permeable antioxidants, or by the adenovirus-mediated intracellular overexpression of Cu,Zn-superoxide dismutase, but not by the exogenous (extracellular) administration of the cell-impermeable antioxidants, superoxide dismutase and/or catalase. This ICAM-1 upregulation was also inhibited by inhibitors of NADH dehydrogenase, cytochrome bc1 complex and NADPH oxidase. However, a measurable increase in net cellular ROM generation in response to TNF-alpha was not seen using four disparate sensitive ROM assays. Moreover, the stimulation of exogenous or endogenous ROM generation did not upregulate ICAM-1, nor enhance ICAM-1 upregulation by TNF-alpha. These findings suggest that an ambient background flux of ROMs, generated intracellularly, but not their net incremental generation, is necessary for TNF-alpha to induce ICAM-1 expression in endothelium in vitro.

Adenoviridae↗

PubMed1998

Pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure elevation.

OBJECTIVE: To characterize the pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure (IOP) elevation and to provide individual patients with the optimal dosage regimen for this drug. METHODS: We studied 17 patients with transient IOP elevation, who were given 62.5-500 mg acetazolamide orally as single or repetitive doses. Plasma acetazolamide concentration and IOP were measured at approximately 1, 3, 5, and 9 h after the last acetazolamide administration. Pharmacokinetics and pharmacodynamics were analyzed by nonlinear mixed-effect modeling using the program NONMEM. RESULTS: The plasma concentration profile of acetazolamide was characterized by a one-compartment model with first-order absorption. The apparent oral clearance was related to the creatine clearance (CCR) which was estimated by the Cockcroft and Gault equation, as follows: 0.0468 x CCR1 x h(-1). The estimated apparent oral volume of distribution, first-order absorption rate constant, and absorption lag time were 0.231 l x kg(-1), 0.821 x h(-1), and 0.497 h, respectively. IOP after oral acetazolamide administration was characterized by an Emax model. The maximal effect in lowering the IOP (Emax) was 7.2 mmHg, and the concentration corresponding to 50% of the maximal effect (EC50) was 1.64 microg x ml(-1). As 70% of Emax was achieved at a plasma concentration of 4 microg x ml(-1), this concentration was considered satisfactory for lowering IOP. The recommended dosage was calculated so that the minimum plasma concentration at steady state exceeded this target concentration; 250 mg t.i.d., 125 mg t.i.d., 125 mg b.i.d., and 125 mg once daily for the patients with CCR values of 70, 50, 30, and 10 ml min(-1), respectively. CONCLUSION: Measuring plasma concentrations of acetazolamide and subsequent pharmacokinetic and pharmacodynamic analyses are useful for estimating its concentration-dependent effectiveness in lowering the IOP in individual patients. The dosage regimen presented in this study is expected to improve the benefits of acetazolamide pharmacotherapy in most elderly patients with transient rises in IOP following intraocular surgery.

Acetazolamide↗

PubMed1998

Rice has two distinct classes of protein kinase genes related to SNF1 of Saccharomyces cerevisiae, which are differently regulated in early seed development.

We have isolated five cDNA clones (osk1-5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2-5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments. Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley, and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation. Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds.

Carbohydrate Metabolism↗

PubMed1998

The inhibitory effect of propranolol on ATP-sensitive potassium channels in neonatal rat heart.

1. Whole cell and single channel recordings of ATP-sensitive K+ current (I(K,ATP)) were carried out in ventricular myocytes isolated from neonatal rat hearts. 2. (+/-)-Propranolol, a commonly used beta-blocker, inhibited the whole cell I(K,ATP) in a concentration-dependent manner with a half-maximal concentration (IC50) of 6.7 +/- 1.4 microM, whereas it blocked the inward rectifier K+ current (I(K,I)) only at much higher concentrations (IC50 = 102.4 +/- 20.2 microM). The inhibition was time- and voltage-independent. 3. In the outside-out patch configuration, (+/-)-propranolol inhibited I(K,ATP) (IC50 = 9.8 +/- 2.9 microM) by decreasing the open probability of the channel without inducing additional noise in the open-channel current or a decrease of single channel conductance. The single channel current of I(K,I) was also blocked by (+/-)-propranolol in the same way as I(K,ATP). 4. (+)-Propranolol, an optic isomer having no beta-blocking effect, inhibited I(K,ATP) (IC50 = 5.8 +/- 1.0 microM), whilst atenolol, a selective beta1-blocker had no effect. Neither GDPbetaS (1 mM) nor GTPgammaS (200 microM) included in the pipette solution modulated the inhibitory effect of (+/-)-propranolol. 5. We concluded that the inhibitory effect of (+/-)-propranolol was not via the beta-adrenergic signal transduction pathway, but by direct inhibition of I(K,ATP) channels.

Adenosine Triphosphate↗

PubMed1998

Expression analysis of the autolysin gene (atl) of Staphylococcus aureus.

The bifunctional autolysin gene (atl) of Staphylococcus aureus was transcribed into a 4.1-kb transcript. The transcription initiation site was located at an adenine residue 33-nt upstream from the putative atl start codon. Analysis using a promoter-reporter plasmid showed that promoter activity increased during the exponential growth phase. The Tn551 insertion site of the autolysis-deficient mutant S. aureus RUSAL2 was located in the putative catalytic region of the glucosaminidase domain.

Base Sequence↗

PubMed1998

Renal excretion of rhodamine 123, a P-glycoprotein substrate, in rats with glycerol-induced acute renal failure.

To clarify renal handling of rhodamine 123, a substrate for P-glycoprotein, in normal and diseased states, in-vivo clearance studies were performed with normal rats and rats with glycerol-induced acute renal failure. For normal rats the excretion ratio of unbound rhodamine 123-to-inulin was 3.25, indicating the presence of the renal tubular secretion of rhodamine 123. Co-administration of cyclosporin, a P-glycoprotein inhibitor, significantly reduced tubular secretion of rhodamine 123. Administration of glycerol induced both an increase in blood urea nitrogen and a reduction in the glomerular filtration rate, confirming the induction of acute renal failure. Total plasma, renal, and tubular secretory clearances of rhodamine 123 were significantly lower for rats with acute renal failure than for control rats. There was no difference between the ATP content of the renal cortex in control rats and those with acute renal failure. In addition to the decrease in renal clearance, a decrease in the biliary clearance of rhodamine 123 was also observed in rats with acute renal failure. These results imply that rhodamine 123 is secreted via P-glycoprotein in renal tubules and that the renal secretory clearance of rhodamine 123 was reduced after acute renal failure, probably because of impairment of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Hepatic clearance of ONO-5046, a novel neutrophil elastase inhibitor, in rats and in the rat perfused liver.

The hepatic clearance of ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulphonylamino]benz oyl]aminoacetic acid), a low-molecular-weight neutrophil elastase inhibitor, has been investigated in rats and in the rat perfused liver. This ester was easily hydrolysed to its inactive metabolite EI-601 (N-[2-[(4-hydroxyphenyl)sulphonylamino]benzoyl]aminoacetic acid) in liver homogenate and in erythrocytes suspension in-vitro. On the other hand, it was stable in biological media such as plasma and whole blood, which contain plasma proteins. Scatchard plot analysis of ONO-5046 binding to bovine serum albumin (BSA) in-vitro indicated that the association constant (K) and number of binding sites (n) were 6.91 x 10(4) (M(-1)) and 4.33, respectively. Thus, ONO-5046 (100 microM) would bind to plasma proteins to an extent >99% at physiological plasma-protein concentrations. The total plasma clearance of ONO-5046 in rats was constant (approximately 9 mL min(-1) kg(-1)) under different steady-state plasma concentrations (5-50 microM) a value equivalent to the hepatic clearance. In the rat perfused liver, the hepatic extraction ratio of ONO-5046 was significantly reduced by adding BSA to the dosing solution. Thus, the relatively low hepatic clearance of ONO-5046, which has an ester linkage in its structure and is naturally susceptible to enzymatic hydrolysis, was found to be because of the extremely high protein-binding of the compound.

Animals↗

PubMed1998

In-vivo calibration of microdialysis probe by use of endogenous glucose as an internal recovery marker: measurement of skin distribution of tranilast in rats.

To estimate the absolute concentration of substrates surrounding a microdialysis probe in-vivo, we developed a simple calibration method using endogenous glucose as an internal recovery marker and determined the skin distribution of tranilast (N-(3,4-dimethoxy-cinnamoyl)anthranic acid), an anti-allergic agent, in rats. This calibration method was based on the assumption that the concentration of glucose in the extracellular fluid of skin tissues is the same as that in plasma and that the in-vivo recovery ratio of glucose to tranilast by microdialysis is the same as that estimated in-vitro. Based on these assumptions, the dialysate concentrations of tranilast and glucose recovered from cutaneous microdialysis, glucose concentration in plasma, and in-vitro recovery ratio of tranilast to glucose by microdialysis were determined for the estimation of absolute unbound concentration of tranilast in the extracellular fluid of skin tissues. In an in-vitro study employing plasma containing tranilast, the unbound concentration of tranilast in plasma estimated from the dialysate concentration was just comparable with that determined by ultrafiltration methods. Also in an in-vivo study under steady-state plasma concentration of tranilast in rats, the estimated concentration of tranilast in the skin extracellular fluid was the same level as the unbound concentration of tranilast in plasma. Using the present calibration method, the skin distribution of tranilast administered into the intestinal loop or transdermally was continuously monitored in a quantitative manner.

Animals↗

PubMed1998

Protective roles of gamma delta T cells and interleukin-15 in Escherichia coli infection in mice.

The number of gamma delta T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The gamma delta T cells preferentially expressed invariant Vgamma6 and Vdelta1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of gamma delta T cells by T-cell receptor delta gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of gamma delta T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated gamma delta T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the gamma delta T cells.

Animals↗

PubMed1998

Expression of low-molecular-weight kininogen in mouse vascular smooth muscle cells.

To determine the existence of the kallikrein-kinin system in vascular wall, the expression of low-molecular-weight (LMW) kininogen, a precursor protein of kinins, was studied in mouse aortic smooth muscle in vivo or in cultured vascular smooth muscle cells (VSMC) derived from mouse aorta in vitro. Although LMW-kininogen mRNA was undetectable in aortic smooth muscle of untreated mice using either Northern blotting or reverse transcription-polymerase chain reactions (RT-PCR) followed by Southern blotting, administration of lipopolysaccharide (LPS; 1 mg/kg, i.v.) induced the expression of LMW-kininogen mRNA at levels detectable by RT-PCR within 12 h. Cultured VSMC not only expressed LMW-kininogen mRNA at levels easily detectable by RT-PCR, but also secreted LMW-kininogen-like protein that was immunoreactive to anti-mouse LMW-kininogen antibody. These results demonstrate that VSMC are a source of LMW-kininogen in the mouse, and suggest the presence of a local kallikrein-kinin system in vascular tissue. LPS-induced up-regulation of LMW-kininogen expression suggests a role for vascular LMW-kininogen in tissue trauma or inflammation.

Animals↗

PubMed1998

Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions.

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.

Amidohydrolases↗

PubMed1998

Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase.

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.

Amidohydrolases↗

PubMed1998

Immobilization of N-carbamyl-D-amino acid amidohydrolase.

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.

Amidohydrolases↗

PubMed1998

Isolation of Agrobacterium sp. strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme.

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coli. The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285. The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells. The enzyme was purified and some properties were investigated. Both the optimum pH and the pH that gave greatest stability were about pH 7.0. The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

Amidohydrolases↗

PubMed1998

Screening, characterization, and cloning of the gene for N-carbamyl-D-amino acid amidohydrolase from thermotolerant soil bacteria.

For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45 degrees C with N-carbamyl-D-amino acids as the sole nitrogen source. The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction. One of the enzymes, produced by Pseudomonas sp. strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced. A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli. The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000. The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp. strain KNK712, was 60%. A database was searched for similar sequences.

Amidohydrolases↗