Biomedical subjects

A Tsuji

Publications and source records attributed to A Tsuji.

At least 19 recordsLinked to original sources

PubMed1992

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Correction of scattered photons in Tc-99m imaging by means of a photopeak dual-energy window acquisition.

We are proposing a new method for correcting of scattered photons in technetium-99m (99mTc) imaging by means of photopeak dual-energy window acquisition. This method consists of the simultaneous acquisition of two images and estimation of a scatter image included in the symmetric energy window (SW) image by the difference between these images. The scatter corrected image is obtained by subtracting the scatter image from the SW image. In order to evaluate this method, we imaged a planar and a SPECT phantom with cold lesions and calculated the contrast value with and without the scatter correction. In addition, we performed asymmetric energy window (ASW) imaging to compare with this scatter correction method for planar images. In the planar image with the tissue-equivalent material of 10 cm, the scatter correction method removed 32% of the counting rate of the SW image and improved from 0.81 to 0.94 of the contrast value for a 4 cm-diameter cold lesion, while the contrast value with the ASW was 0.87 for such a cold lesion. The scatter corrected SPECT image had a reduction of 18% of the counting rate of the SW SPECT image and improvement of approximately 11% in contrast for cold spot sizes larger than a 3 cm-diameter, compared with the SW SPECT image. In addition, a perfusion defect could be well visualized by this scatter correction method on 99mTc-HMPAO regional cerebral blood flow SPECT of a patient. Our proposed scatter correction method can improve both planar and SPECT images qualitatively and quantitatively.

Brain Concussion↗

PubMed1992

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells.

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells.

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)2NH2), was examined in cultured monolayers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM-1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 +/- 3.27 pmol/mg of protein with a half-saturation constant (Kd) of 15.9 +/- 6.4 microM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.

Adrenocorticotropic Hormone↗

PubMed1992

In-vivo blood-brain barrier transport of a novel adrenocorticotropic hormone analogue, ebiratide, demonstrated by brain microdialysis and capillary depletion methods.

The transport of ebiratide, a novel adrenocorticotropic hormone (ACTH) analogue, [H-Met-(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8-NH2], through the blood-brain barrier was directly demonstrated in-vivo. [125I]Ebiratide (16.9 MBq mL-1) or [14C]sucrose (29.2 MBq mL-1) known to be restrictively transported through the blood-brain barrier was infused into the rat internal carotid artery at a flow rate of 50 microL min-1 for 10 min, and after 15 min infusion the distribution volume of each compound in the brain parenchyma was determined by the capillary depletion method. The distribution volume of [125I]ebiratide was 167.8 +/- 62.2 microL (g brain)-1, which was about seven times higher than that of [14C]sucrose (24.9 +/- 4.0 microL g brain)-1, indicating the uptake of ebiratide into brain parenchymal cells. During the infusion into the internal carotid artery, brain microdialysis was simultaneously performed to directly collect the brain interstitial fluid as the dialysate. Radioactivity was detected in the dialysate during the [125I]ebiratide infusion and HPLC analysis of the dialysate revealed that the intact ebiratide accounted for greater than or equal to 80% total radioactivity. The concentrations of [125I]ebiratide and [14C]sucrose in the brain interstitial fluid were estimated based on the relative recovery obtained in the in-vitro recovery study. The brain interstitial fluid/internal carotid arterial blood concentration ratio for [125I]ebiratide was determined to be 1.47 x 10(-2) +/- 0.17 x 10(-2) and was about eight times higher than that for [14C]sucrose (1.92 x 10(-3) +/- 0.36 x 10(-3)), indicating significant transport of ebiratide to the brain interstitial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1992

[The study of pathogenic mechanisms of chronic Pseudomonas aeruginosa lung infections by mucoid strains].

Chronic Pseudomonas aeruginosa lung infection with mucoid strains is the predominant cause of death in cystic fibrosis (CF) or diffuse panbronchiolitis (DPB). This infection is characterized by a chronic course without spread of the bacteria to the blood when compared with other infections due to the non-mucoid strains. However, the mechanism of P. aeruginosa lung infection with the mucoid strains remains obscure. Intra-tracheal and systemic infection in mice, susceptibility to the bactericidal activity of fresh human and mouse serum, and adherent activity to mouse fetal lung cell were examined for mucoid and non-mucoid strains of P. aeruginosa. After intra-tracheal infection, the mucoid strains were distributed to other organs anormously but not the non-mucoid stains, and the bacterial number of the mucoid strains in the blood were significantly lower than that of the non-mucoid strains. On the other hand, when these strains were inoculated into the tail vein of mouse, the mucoid strain was eliminated more rapidly from blood as compared with the non-mucoid strain. The mucoid strains showed reduced bacteremic virulence when compared with non-mucoid strains with a 50% lethal dose (LD50) of 1.5 x 10(7) CFU/mouse as the mean value in a systemic infection. In contrast to the non-mucoid strains, the mucoid strains were sensitive to human fresh serum but were resistant to mouse fresh serum. The mucoid strains adhered to the monolayer of the mouse fetal lung cell 7-fold better than did non-mucoid strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Mechanisms of intestinal absorption of the antibiotic, fosfomycin, in brush-border membrane vesicles in rabbits and humans.

In order to clarify the mechanism of intestinal absorption of an antibiotic, fosfomycin (FOM), the uptakes of FOM by rabbit and human small intestinal brush-border membrane vesicles (BBMV) were studied. The initial uptake of FOM by BBMV at 15 s was saturable at a higher concentration of FOM. The kinetic parameters at 37 degrees C of the saturable uptake expressed by the Michaelis-Menten equation were Kt = 5.17 mM and Jmax = 3.88 nmol/15 s/mg protein for rabbits, and Kt = 4.03 mM and Jmax = 1.90 nmol/15 s/mg protein for humans. The most efficient uptake was observed in the presence of both inward-directed Na(+)- and H(+)-gradients in both mammals. The uptake of FOM was inhibited by inorganic phosphate, FOM glycol which is a degradation product of FOM in the gastric juice and specific inhibitors of phosphate transport such as arsenate and phosphonoacetic acid. These findings confirmed that FOM absorption from rabbit and human small intestines is associated with the phosphate transport system. These transport phenomena of FOM are in close agreement with those obtained previously in rat BBMV studies. Judging from the results obtained for three mammalian species, rat, rabbit and human, it was concluded that carrier-mediated transport via the phosphate transport system is a very important pathway of intestinal absorption of FOM.

Animals↗

PubMed1992

An application of microdialysis to drug tissue distribution study: in vivo evidence for free-ligand hypothesis and tissue binding of beta-lactam antibiotics in interstitial fluids.

To prove the free-ligand hypothesis for extravascular equilibration and tissue binding mechanism of beta-lactam antibiotics, the microdialysis technique has been employed for the lung, the muscle and the liver in rats. Cefminox, a cephem antibiotic, and SY5555, a new penem antibiotic, were used in the present study. During the constant infusion of each antibiotic with simultaneous infusion of antipyrine, the microdialysis studies were performed and the dialysate concentrations were determined. The dialysate concentration was extrapolated to the in vivo unbound concentration in tissue interstitial fluids (Cisf,u) according to the extrapolation method which was derived from the clearance concept. This extrapolation method incorporates the effective dialysis coefficient of a reference compound, antipyrine, which is used to correct the difference between in vivo and in vitro permeabilities of microdialysis fiber. The values of Cisf,u values for cefminox and SY5555 in the lung, muscle and liver were close to the unbound concentrations in the venous plasma leaving these organs. Furthermore, good coincidences were obtained between the unbound concentrations of SY5555 in lung and muscle interstitial fluids estimated from the total concentrations in homogenized tissues and those extrapolated by the microdialysis studies. Consequently, the present microdialysis studies provided the in vivo evidence that 1) the free-ligand hypothesis for extravascular equilibration of beta-lactam antibiotics is true, and that 2) beta-lactam antibiotics are restricted in the interstitial space in a noneliminating organ and bind only with albumin existing in this space.

Animals↗

PubMed1992

Selective analysis of mutual displacement effects at the primary binding sites of phenoxymethylpenicillin and cephalothin bindings to human serum albumin.

In order to analyze the mutual displacement effects on the protein binding of beta-lactam antibiotics, binding experiments with the human serum albumin (HSA) were performed for cephalothin (CET) and phenoxymethylpenicillin (PCV) by using the centrifugal ultrafiltration method. The numbers of primary and secondary binding sites, n1 and n2, and the affinity constants for the primary and secondary binding sites, K1 and K2 were determined for CET to be 1.00 +/- 0.06 (mean +/- S.D.) and 4.54 +/- 0.12 and 2.59 x 10(3) +/- 0.10 x 10(3) (M-1) and 2.59 x 10(2) +/- 0.16 x 10(2) (M-1), respectively, and for PCV to be 0.94 +/- 0.10 and 5.41 +/- 0.40 and 3.52 x 10(3) +/- 0.25 x 10(3) (M-1) and 4.07 x 10(2) +/- 0.54 x 10(2) (M-1), respectively. Using the predicted optimum unbound concentration of PCV, i.e., 4.6 x 10(-4) M, the displacement effect of PCV to the binding of CET at the primary site has been demonstrated, while no significant effect was observed at the secondary binding site. Moreover, a competitive displacement effect of CET was also demonstrated for the binding of PCV to HSA at the primary binding site, suggesting that CET and PCV bound to HSA at the same primary binding site.

Binding Sites↗

PubMed1992

Relationship between lipophilicity and binding affinity with human serum albumin for penicillin and cephem antibiotics.

Relationship between structure and binding affinity to human serum albumin (HSA) has been studied for penicillin and cephem antibiotics. For penicillin analogs, a good correlation between the apparent affinity constants, Kapp, for HSA binding and the partition coefficient, Papp, determined in isobutyl alcohol-pH 7.4 phosphate buffer system was observed, indicating that the hydrophobic interaction of 6-substituent of penicillins with amino acid of HSA would play an important role for the binding. However, no correlation between the Kapp and Papp values was observed for cephem antibiotics. Mutual competitive displacement effects were demonstrated for the primary binding sites of cephalothin, cefazolin, cefotetan and cefatrizine, suggesting the presence of a common binding region in HSA among these cephem antibiotics examined. Significant differences were observed for the Kapp value among cephems having the same 3-substitute of N-methylthiotetrazole in the molecule, i.e., cefpiramide, cefotetan, cefoperazone, cefamandole, cefmenoxime, cefmetazole and cefbuperazone, suggesting that 7-substitute of cephem would play an important role for the binding with HSA. Moreover, comparing the binding affinity and the structure of 3-substitute for cephems, all of the analogs having a heterocycle bind strongly with HSA in spite of their low lipophilicity. These observations suggest that an interaction between heterocycle at the position 3 and HSA would contribute to an additional binding force for the binding of cephem antibiotics to HSA.

Anti-Bacterial Agents↗

PubMed1992

[The evidence of safety of immunotherapy for unexplained habitual aborters].

Immunotherapy (immunization twice at 5 and 7 weeks of gestation with X-irradiated 200ml total blood or 1-200 x 10(6) mononuclear cells from the husband) was performed on 200 women without autoimmune abnormalities but experiencing habitual abortions of unknown causes between August, 1982 and September, 1990. One hundred fifty-seven babies were eventually born alive. The oldest offspring was 7 yrs 4 mos in August, 1991. We investigated the safety of the immunotherapy in terms of its effects on the women, conditions during pregnancy/labor and conducted long-term follow-up on offspring. Among 190 women whose pregnancy terminated by September, 1990, 152 cases gave birth, including 5 sets of twins. The successful rate was 80.0%. There were no complications among the 200 cases, only 2 small-for-dates babies (1.3%) among 157 babies born alive, and 1 baby born dead in the 3rd trimester, but that was not directly related with immunotherapy. Tsumori-Inage Infant Mental Development Test found no abnormalities (3 yrs and over). At 3 yrs, and only 1 child scored under the mean--2 standard deviation in height and weight. Thus, the safety of the immunotherapy was confirmed by long-term follow-up of both mother and child in which no notable complications resulting from immunotherapy with the husband's X-irradiated cells could be found.

Abortion, Habitual↗

PubMed1992

[Usefulness of magnetic resonance imaging (MRI) in evaluation of left ventricular apical aneurysm].

Twenty three consecutive cases of left ventricular aneurysm due to antero-septal myocardial infarction in normal sinus rhythm were studied to decide whether or not magnetic resonance imaging (MRI) can evaluate aneurysm of the left ventricular apex. The apex, as well as the base, of the left ventricle was clearly imaged in 21 out of 23 cases. Poor images were obtained in two cases who showed frequent premature ventricular beats during this procedure. Two-dimensional echocardiography has weak points when visualizing left ventricular apex because, for one thing, the sector angle is maximally 90 degrees, and for another, the cardiac apex is too near the probe for observation of the characteristic of the left ventricular apical wall. However, MRI can make up for the above weak points and can provide supportive information by visualizing the entire left ventricular apex.

Adult↗

PubMed1991

Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography.

A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5'-nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher (P less than 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5'-nucleotidase deficiency.

5'-Nucleotidase↗

PubMed1991

Hepsin, a cell membrane-associated protease. Characterization, tissue distribution, and gene localization.

Hepsin, a putative membrane-bound serine protease, was originally identified as a human liver cDNA clone (Leytus, S.P., Loeb, K.R., Hagen, F.S., Kurachi, K., and Davie, E.W. (1988) Biochemistry 27, 1067-1074). In the present study the human hepsin gene was localized to chromosome 19 at q11-13.2. The messenger RNA of hepsin is 1.85 kilobases in size and present in most tissues, with the highest level in liver. Hepsin is synthesized as a single polypeptide chain, and its mature form of 51 kDa was found in various mammalian cells including HepG2 cells and baby hamster kidney cells. It is present in the plasma-membrane in a molecular orientation of type II membrane-associated proteins, with its catalytic subunit (carboxyl-terminal half) at the cell surface, and its amino terminus facing the cytosol. Hepsin is found neither in cytosol nor in culture media. The results obtained suggest that hepsin has an important role(s) in cell growth and function.

Amino Acid Sequence↗

PubMed1991

Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells.

A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.

Animals↗

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↗