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Biomedical subjects

A Tsuji

Publications and source records attributed to A Tsuji.

At least 307 records · Page 17Linked to original sources
PubMed1991

The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha 2-macroglobulin and thrombin.

A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.

Amino Acid Sequence↗
PubMed1991

Receptor-mediated endocytosis of A14-125I-insulin by the nonfiltering perfused rat kidney.

The mechanism of insulin uptake and/or degradation in the peritubular circulation of the kidney was investigated using nonfiltering perfused rat kidneys, in which glomerular filtration was sufficiently reduced. After perfusion of A14-125I-insulin in the nonfiltering kidney for designated intervals, the acid-wash technique was employed to separately measure the acid-extractable and acid-resistant A14-125I-insulin, which were quantitated by HPLC and TCA-precipitability. HPLC profiles showed that the nonfiltering kidney metabolizes A14-125I-insulin only to a small extent during 1-h perfusion, suggesting that the peritubular clearance of A14-125I-insulin was not due to extracellular degradation but for the most part to uptake by the kidney. Acid-extractable A14-125I-insulin rapidly increased with time and reached pseudo-equilibrium with perfusate at approx. 10 min, whereas acid-resistant A14-125I-insulin increased continuously. An endocytosis inhibitor, phenylarsine oxide, inhibited significantly the acid-resistant A14-125I-insulin with no change in acid-extractable A14-125I-insulin, suggesting that the peritubular uptake of A14-125I-insulin largely represents endocytosis of the peptide into the intracellular space. Moreover, both the acid-extractable and acid-resistant A14-125I-insulin were significantly decreased in the presence of unlabeled insulin (1 microM). These lines of evidence suggest that insulin is taken up by the nonfiltering perfused kidney via receptor-mediated endocytosis (RME), which possibly occurs at the basolateral side of renal tubular cells, and that the peritubular clearance of insulin is largely accounted for by this mechanism.

Animals↗
PubMed1991

Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency.

The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1991

The reduction of nonspecific binding in chemiluminescent sandwich enzyme immunoassays.

The reduction in nonspecific binding obtained in EIA procedures based on glucose oxidase (GO)-labelled antibody is determined by the origin of the antibodies used for both the solid phase and for GO labelling. We have examined the relationship between nonspecific binding and various antibody combinations for the purpose of establishing highly sensitive chemiluminescent sandwich enzyme immunoassays. It was shown that nonspecific binding could be reduced by the following combination of solid phase and GO labelled antibodies: (i) guinea pig IgG-guinea pig IgG, (ii) goat IgG-rabbit IgG, (iii) goat IgG-rabbit Fab'. On the basis of these results, we succeeded in establishing highly sensitive chemiluminescent sandwich enzyme immunoassays for human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP). The detection limit was 2.5 mIU/ml for hCG using combination (i), 1 ng/ml for AFP using combination (ii), and 0.05 ng/ml (70 amol/assay) for AFP using combination (iii).

Animals↗
PubMed1991

Receptor-recycling model of clearance and distribution of insulin in the perfused mouse liver.

After perfusion of mouse livers with A14-125I-insulin for designated intervals, an acid-wash technique was employed to separately measure the surface-bound (Xs) and intracellular (Xi) A14-125I-insulin, as well as intracellular degradation products (Xdeg) of labelled insulin. From the perfusate concentrations (Cp) of A14-125I-insulin, the apparent intrinsic hepatic clearance of labelled insulin at a high dose (0.2 nmol/l) was shown to be 60% smaller than that at a low dose (0.018 nmol/l), indicating that the cellular uptake of insulin is remarkably nonlinear at the concentration range examined. From the time courses of Cp, Xs, Xi and Xdeg, the hepatic insulin disposition was shown to be largely accounted for by the receptor-mediated endocytosis. The observed data at the low dose were analysed to estimate biochemical parameters, (i.e., total receptor number, endocytotic rate constant and intracellular degradation rate constant) according to "receptor-recycling" and "non-receptor-recycling" models, using a computer-aided optimization procedure. The "receptor-recycling" model could not only adequately explain the Cp, Xs, Xi and Xdeg at the low dose, but also predict the Cp at the high dose. On the other hand, a "non-receptor-recycling" model, in which recycling of receptors was not assumed, could also explain the observed data at the low dose, but failed to predict the Cp at the high dose, indicating that the receptor recycling process is necessary to explain the hepatic insulin clearance at high insulin concentrations, at which hepatic insulin clearance should be limited by the rate of receptor recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applications to enzyme immunoassay and DNA probe assay.

Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H2O2). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), beta-D-galactosidase (beta-gal), and beta-glucosidase were assayed by this method using 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and 5-bromo-4-chloro-3-indolyl-beta-D-glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10(-19) mol of ALP and beta-gal, respectively. This assay system can be applied to enzyme immunoassay and DNA probe assay.

Alkaline Phosphatase↗
PubMed1991

In vivo transport of a dynorphin-like analgesic peptide, E-2078, through the blood-brain barrier: an application of brain microdialysis.

In vivo transport through the blood-brain barrier (BBB) has been demonstrated for a dynorphin-like analgesic peptide, CH3-[125I]Tyr-Gly-Gly-Phe-Leu-Arg-CH3Arg-D-Leu-NHC2H5 ( [125I]E-2078). A remarkable time-dependent increase in the distribution volume of [125I]E-2078 in the brain parenchyma separated from blood vessels and capillaries was observed during a brain perfusion. The distribution volume of [125I]E-2078 in the brain parenchyma after 20 min of perfusion was 2.18 +/- 0.09 microliters/g brain (mean +/- SE) and was significantly greater than the distribution volume of [3H]inulin (0.994 +/- 0.138 microliters/g brain), providing in vivo evidence for the penetration of [125I]E-2078 into the brain parenchyma. Brain microdialysis was carried out to collect directly the brain interstitial fluid (ISF) during the brain perfusion of [125I]E-2078. No metabolite of [125I]E-2078 in the brain ISF was found by high-performance liquid chromatographic analysis of the brain dialysate. The concentrations of [125I]E-2078 and [14C]sucrose in the brain ISF were estimated based on an in vitro evaluation of dialysis clearance. The concentration ratio of [125I]E-2078 between the brain ISF and the brain perfusate was determined to be 2.92 x 10(-1) +/- 0.50 x 10(-1) and was approximately 100 times higher than that of [14C]sucrose (2.71 x 10(-3) +/- 1.43 x 10(-3), demonstrating transport of [125I]E-2078 through the BBB in vivo. On the other hand, no remarkable difference in the cerebrospinal fluid (CSF)-to-perfusate concentration ratios of [125I]E-2078 and [14C]sucrose was observed, indicating little contribution of the blood-CSF barrier (BCSF barrier) transport to the penetration of [125I]E-2078 into the brain.

Amino Acid Sequence↗
PubMed1991

Effect of receptor up-regulation on insulin pharmacokinetics in streptozotocin-treated diabetic rats.

The present study investigated the mechanism by which the disposition of insulin is altered in streptozotocin (STZ)-treated diabetic rats as compared with 48-hr-fasted normal (control) rats. It was shown by an indocyanine green infusion method that the hepatic plasma flow rate (QH) in diabetic rats (1.64 ml/min/g liver) is significantly higher than that in control rats (0.982 ml/min/g liver). The portal injection technique revealed that the unidirectional clearance (CLon), which represents the binding of A14-125I-insulin to surface receptors in the liver, is significantly elevated in diabetic rats, suggesting an increase in the surface receptor number (RT), i.e., up-regulation in the liver. In both control and diabetic rats, the total-body clearance (CLtot) and steady-state volume of distribution (Vdss) of labeled insulin decreased significantly with a simultaneous injection of unlabeled insulin (8 U/kg), confirming that the disposition of insulin is affected largely by specific, saturable receptor-mediated processes. The CLtot and Vdss increased significantly in diabetic rats, while nonspecific portions of these parameters were not changed. From the increases in CLtot (80%) and QH (67%) in diabetic rats, a pharmacokinetic analysis has revealed a 40% increase in the hepatic intrinsic clearance (CLint,sp) of A14-125I-insulin via a specific mechanism in diabetic rats. In conclusion, we have provided in vivo evidence for a small increase in CLint,sp of insulin in STZ-diabetic rats compared with control rats, which may be caused by an increase in the surface receptor number in the livers of diabetic rats.

Animals↗
PubMed1991

In-vitro evidence for carrier-mediated uptake of acidic drugs by isolated bovine brain capillaries.

[3H]Acetic acid was taken up into the isolated brain capillaries in a temperature and concentration dependent manner. The initial uptake rates were pH dependent over the range 5.0-7.4. An increase of the uptake rate with decrease in medium pH was not seen in the presence of unlabelled acetic acid (10 mM). The uptake of [3H]acetic acid was significantly reduced in the presence of several drugs having a monocarboxylic group. The initial uptake of [14C]salicylic acid was also pH dependent and was inhibited by acetic acid and acidic drugs. A mutually competitive inhibition was observed for the uptake of acetic acid and salicylic acid. The present study demonstrates that acidic drugs having a monocarboxylic group are transported by a common carrier-mediated system into the brain capillaries.

Acetates↗
PubMed1991

[Effect of macrolide antibiotics on human serum-bactericidal sensitivity of Pseudomonas aeruginosa S-6].

It is well known that long-term administration of erythromycin (EM) at a small dose is effective for persistent infections with Pseudomonas aeruginosa in diffuse panbronchiolitis or chronic bronchitis patients. Since EM is less active against P. aeruginosa in vitro, we have been interested in the mechanisms of clinical efficacy of EM in these patients. This study examines the effect of macrolide antibiotics on human serum-bactericidal sensitivity of P. aeruginosa S-6, clinically isolated from the patient with respiratory tract infection. A significant increase in serum-bactericidal sensitivity of P. aeruginosa S-6 was observed on agar containing EM of 10 micrograms/ml after incubation for 36-60 hours (p less than 0.05). The enhancement of serum sensitivity of P. aeruginosa S-6 was apparently observed even at a concentration of EM 1.5 micrograms/ml after the 48 hours incubation (p less than 0.01). Of other macrolide antibiotics used, clarithromycin (CAM) also increased the serum-bactericidal sensitivity of P. aeruginosa S-6 as well as EM, however no change in the sensitivity was found with kitasamycin, josamycin, rokitamycin and oleandomycin. The results suggest that the change of serum-bactericidal sensitivity of P. aeruginosa induced by EM or CAM may, in part, contribute to the clinical efficacy of these antibiotics against persistent pulmonary P. aeruginosa infections.

Anti-Bacterial Agents↗
PubMed1991

In vitro antibacterial activity of ME1207, a new oral cephalosporin.

ME1207 is the prodrug of ME1206. Its in vitro antibacterial activity was compared with that of cefteram, cefpodoxime, cefixime, and cefaclor against various clinical isolates. ME1206 was more active than the other cephems tested against staphylococci, streptococci, Morganella morganii, Pseudomonas cepacia, and Flavobacterium meningosepticum and had the most potent activity against Haemophilus influenzae and Neiserria gonorrhoeae. The drug also showed a wide spectrum of activity against other gram-positive and gram-negative bacteria, except methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Citrobacter freundii, Pseudomonas aeruginosa, Xanthomonas maltophilia, and Alcaligenes xylosoxydans.

Administration, Oral↗
PubMed1991

Transport of the new quinolone antibacterial agents of lomefloxacin and ofloxacin by rat erythrocytes, and its relation to the nicotinic acid transport system.

Transport mechanism of quinolonecarboxylic acid derivatives (quinolones), lomefloxacin and ofloxacin, in rat erythrocytes was investigated. The uptake of lomefloxacin by erythrocytes was apparently nonsaturable over the concentration range of 0.1-1.0 mM at 27 degrees C and pH 7.4. In the case of ofloxacin, however, the erythrocytes took up ofloxacin in a concentration dependent manner at 20 degrees C and pH 7.4, with a maximum rate (Jmax) of 28.8 +/- 0.98 pmol/10(6) cells/s and a Michaelis constant (Kt) of 0.92 +/- 0.15 mM. An increase in the medium osmolarity caused a decrease in the [14C]lomefloxacin uptake, indicating that the drug is transported into the intracellular space of erythrocytes. The uptake of [14C]lomefloxacin by the erythrocytes was independent of pH but increased above pH 6.0, and, moreover, was dependent on temperature with an activation energy of 16.6 kcal/mol. The uptake of [14C]lomefloxacin was competitively inhibited by a water-soluble vitamin, nicotinic acid, with an inhibition constant (Ki) of 16.1 mM, but not inhibited by 10 mM of L-alanine, glucose, p-aminohippuric acid (PAH), N-methyl-nicotinamide (NMN) or tetraethylammonium chloride (TEA). These lines of evidence suggest that the transport system of lomefloxacin and ofloxacin in rat erythrocytes is common with that of a water-soluble vitamin, nicotinic acid, to which quinolones are structurally analogous.

Animals↗
PubMed1991

Muscle microdialysis as a model study to relate the drug concentration in tissue interstitial fluid and dialysate.

The steady-state dialysis kinetics in buffer, erythrocyte suspension and muscle have been analyzed by clearance theory in the microdialysis study. "Tube" model has been demonstrated to be a useful model to relate the dialysis clearance, CLD, the dialysis flow rate, F, and the permeability rate constant, PA, for microdialysis employing the transcranial type microdialysis probe. The effective dialysis coefficient (Rd), defined as the ratio of the in vivo PA and in vitro PA, was introduced to account for the differences between in vivo and in vitro microdialyses. The Arrhenius plot of the antipyrine permeability rate constant presented a single straight line in the range of 15-37 degrees C with an activation energy of 5.49 kcal/mol. A fairly good correlation was observed between the reciprocal of the permeability rate constant and the root of the molecular weight in the range of 18-1039. On the contrary, the molecular weight and the plasma membrane permeability were not determinant factors for Rd value determined in the erythrocyte suspension (Rd,erythrocyte), while the interstitial fluid space (100-hematocrit)% of erythrocyte suspension plays a dominant factor to change Rd,erythrocyte. The in vivo permeability rate constant was determined in the muscle for [3H]water, [14C]urea, antipyrine and [14C]sucrose under the steady-state condition. No significant difference of Rd in muscle tissue was demonstrated for these four model substances. By using the Rd value, a hypothetical equation has been proposed to relate the concentration in the dialysate and the interstitial fluid at steady-state.

Animals↗
PubMed1991

Participation of monocarboxylic anion and bicarbonate exchange system for the transport of acetic acid and monocarboxylic acid drugs in the small intestinal brush-border membrane vesicles.

A participation of bicarbonate exchange system for the transport of acetic acid and the related monocarboxylic acid drugs in the intestinal brush-border membrane vesicles (BBMVs) was investigated. The uptake of [3H]acetic acid at 37 degrees C by BBMVs was markedly stimulated and showed a clear overshoot phenomenon in the presence of outward-directed bicarbonate gradient (pHin = 7.5, [KHCO3]in or [NaHCO3]in = 100 mM; pHout = 7.5, [K-gluconate]out or [Na-gluconate]out = 100 mM). This uptake process was saturable (Kt = 50.4 +/- 4.96 mM and Jmax = 11.6 +/- 0.61 nmol/mg protein/10 s) and was inhibited by DIDS (4,4-diisothiocyano-2,2'-disulfonic acid stilbene disodium salt) and furosemide, anion exchange inhibitors, and by many monocarboxylates. The initial uptake of [3H]acetic acid was competitively inhibited by salicylic acid, suggesting the common transport between acetic acid and salicylic acid. At lower extravesicular pHs and in the presence of outward-directed bicarbonate gradient (pHin = 7.5 [KHCO3]in = 100 mM; pHout = 6.0 or 5.0, [K-gluconate]out = 100 mM) where membrane potential was clamped to zero by K(+)-valinomycin, the uptake of [3H]acetic acid showed an overshoot phenomenon, whereas the uptake was significantly decreased in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a protonophore. It was concluded, therefore, that there are one or two mechanisms for the carrier-mediated transport of acetic acid and monocarboxylates related to bicarbonate exchange systems in rabbit intestinal BBMVs: 1) proton gradient independent and bicarbonate exchange system; 2) proton gradient dependent and bicarbonate exchange system.

Acetates↗
PubMed1991

Transport mechanism of a new behaviorally highly potent adrenocorticotropic hormone (ACTH) analog, ebiratide, through the blood-brain barrier.

The binding and internalization of a novel adrenocorticotropic hormone (ACTH) analog having a potent neuromodulating effect, ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), by isolated bovine brain capillaries, were examined. Metabolism of [5-125I-His]ebiratide occurred during a 30-min incubation with bovine brain capillaries at 37 degrees C. In the presence of 20 mM EDTA, added to inhibit this metabolism, the medium, after 30 min of incubation, contained 82.3 +/- 0.5% of the unchanged ebiratide. The total binding and acid-resistant binding of [125I]ebiratide increased with time and reached an equilibrium at about 15 min. The total binding and acid-resistant binding at 30 min (as the cell/medium ratios corrected with [14C]sucrose) were 13.07 +/- 0.86 and 5.00 +/- 0.18 microliters/mg of protein, respectively. The acid-resistant binding showed significant dependence on temperature and medium osmolarity. The [125I]ebiratide binding was significantly inhibited by dansylcadaverine, an endocytosis inhibitor. The saturable acid-resistant binding was obtained by the addition of unlabeled ebiratide (100 nM-5 mM), and the maximal internalization capacity (Bmax) at 30 min was 144.2 pmol/mg of protein, with the half-saturation constant (KD) of 62.1 microM. The nonsaturable acid-resistant binding [cell/medium ratio in the presence of the unlabeled compound (1 mM or more)] was 2.2 microliters/mg of protein. The acid-resistant binding was significantly inhibited by human ACTH, poly-L-lysine, protamine and E-2078, a basic peptide, but was not inhibited by poly-L-glutamate, insulin or transferrin. These results demonstrate that ebiratide is transported through the blood-brain barrier via a basic peptide-specific absorptive-mediated endocytosis.

Adrenocorticotropic Hormone↗
PubMed1991

[The changes in cell population and chemical mediators in BALF following local allergen challenge].

In eight subjects who showed dual asthmatic response (DAR) in bronchial provocation tests (BPT) with specific allergens, local allergen challenge (LAC) was conducted using a flexible bronchofiberscope. We examined the concentrations of histamine, leukotriene B4 and C4 (LTB4 and LTC4) and cell populations in bronchoalveolar lavage fluid (BALF) collected before LAC (control), during the immediate response phase (IR), and the late response phase (LR). Control BALF was collected from the left lingula (B4 or B5), and BALF in the IR or LR phase from the right middle lobe (B4 or B5). Each BAL was conducted with 50 ml of saline at 37 degrees C and repeated three times in succession. It was noted that histamine increased significantly (p less than 0.05) in IR-BALF from the control level. In addition, the concentrations of LTC4 and the numbers of eosinophils increased in IR-BALF. In LR-BALF, the numbers of eosinophils (p less than 0.01), and the concentrations of histamine (p less than 0.05), LTC4 (p less than 0.05) and LTB4 increased. From these results, it was suggested that infiltration of eosinophils and various chemical mediators in the bronchial mucosa play important roles in the development of late asthmatic response.

Adolescent↗