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Biomedical subjects

A Tsuji

Publications and source records attributed to A Tsuji.

At least 163 records · Page 9Linked to original sources
PubMed1997

Developmental expression of a novel Kexin family protease, PACE4E, in the rat olfactory system.

PACE4 is a mammalian Kexin family protease that is involved in the maturation of precursor proteins. Four PACE4 isoforms have been identified. We identified a novel PACE4 isoform, PACE4E, from a human cerebellum cDNA library, which possesses a hydrophobic cluster in its C-terminus participating in membrane association. The size of PACE4E mRNA from adult rat brain was estimated by Northern blotting to be 4.4 kb. In situ hybridization histochemistry revealed that the highest level of PACE4E mRNA was expressed in the mitral cells of the adult rat olfactory bulb (OB). The OB is a unique sensory organ in that it has a lifelong regenerating capacity and it affects brain development. We further analyzed the expression of PACE4E mRNA in the developing olfactory system. On day 13.5 of gestation, PACE4E mRNA was expressed at high levels in the neuroepithelium of the forebrain vesicle (FV), olfactory epithelium, and cells in the fiber bundles projecting to the FV. As development proceeded, PACE4E mRNA was expressed in developing mitral cells but decreased in the olfactory epithelium. In the newborn, its expression was confined to the mitral cells in both the main and accessory OB and in some periglomerular cells, as shown in adult rats. The spatio-temporal expression of PACE4E suggests that it plays a role in the establishment and maintenance of the olfactory receptor system.

Animals↗
PubMed1997

Analysis of heart rate variability demonstrates effects of development on vagal modulation of heart rate in healthy children.

OBJECTIVES: Analysis of heart rate variability (HRV) has been found to be a useful method of assessing cardiovascular autonomic control, but normal values for standard HRV measures in children have not been established. We analyzed HRV in 60 healthy children aged 3 to 15 years to determine normal values and to assess the effects of development on cardiac autonomic control with the use of ambulatory electrocardiographic monitoring. RESULTS: The high-frequency (HF) component, an index of cardiac autonomic tone, increased significantly with age from 3 to 6 years (p < 0.01) and decreased with age from 6 to 15 years (p < 0.01), and the magnitude of HF correlated significantly with the R-R intervals. Thus the changes in cardiac autonomic tone could be described as a simple equation using age and heart rate. CONCLUSIONS: We present normal values and changes in the cardiac autonomic system during childhood after HRV analysis, which could lead to a better understanding and treatment of cardiac disease in children.

Adolescent↗
PubMed1997

Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel peptide hormone and has a variety of biological action. In studies of the physiological behaviour of endogenous PACAP, the determination of PACAP levels in biological materials require a highly sensitive and specific method. Therefore, we developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) for PACAP27 which is the biologically important fragment of PACAP. Accordingly, we developed TR-FIA using a biotinylated PACAP27 (b-PACAP27) as a tracer and europium (III) chelate labeled streptavidin-biotinylated bovine serum albumin complex as a detection of biotin on solid phase. A measurable range of PACAP27 was 7.8-1000 pg m1-1 by the proposed TR-FIA. For measurement of biological samples, the samples were purified to eliminate substances which interfered with the TR-FIA. The mean recovery of PACAP27 using commercially reversed phase column was 74.7% (n = 12). The various tissues, extracts and plasma concentrations of rat could be measured by the proposed TR-FIA.

Animals↗
PubMed1997

Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection.

Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.

Codon↗
PubMed1997

A novel human PACE4 isoform, PACE4E is an active processing protease containing a hydrophobic cluster at the carboxy terminus.

PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.

Amino Acid Sequence↗
PubMed1997

Genomic organization and alternative splicing of human PACE4 (SPC4), kexin-like processing endoprotease.

PACE4 (paired basic amino acid cleaving enzyme) is a member of a family of the mammalian kexin-like proprotein convertases containing a subtilisin-like catalytic domain. Previously we reported seven isoform mRNAs of PACE4 that vary in size and 3'-coding sequence [A. Tsuji et al. (1994) Biochem. Biophys. Res. Commun. 200, 943-950; K. Mori et al. (1997) J. Biochem. 121, 941-948]. To determine the origin of these isoforms, the entire human PACE4 gene has been isolated as a set of overlapping genomic DNA fragments, and analyzed by restriction enzyme digestion and nucleotide sequence determination. The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in size from 39 to 1,422 base pairs. Human PACE4 gene is the largest kexin-like proprotein convertase gene reported to date. The most striking feature of its genomic structure is the size of the introns and the number of exons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very similar to that reported for other kexin-like protease genes. The structural analysis of PACE4 genomic DNA indicates that multiple PACE4 transcripts are produced as a consequence of alternative RNA splicing events, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites. A major transcriptional start site was detected 314 bp upstream from the ATG translational start site by primer extension analysis. Sequence analysis of the 5'-flanking region revealed that PACE4 gene lacks TATA and CCAAT boxes in the proximal upstream region of the start site, although potential binding sites for several transcription factors including SP1, AP1, AP2, PEA3, Ets-1, GHF (growth hormone factor)-1, CREB (cyclic AMP response element binding protein), and basic helix-loop-helix proteins, were present. An unusual sequence of six tandem repeats of a nonadecamer (GGCCTGGGGGTTCACCTGC) containing an E box is found in the 5'-flanking region. These results suggest that PACE4 is not a constitutive gene product and its expression is regulated by various transcription factors.

Alternative Splicing↗
PubMed1997

Application of muscle microdialysis to evaluate the concentrations of the fluoroquinolones pazufloxacin and ofloxacin in the tissue interstitial fluids of rats.

Muscle microdialysis has been used to determine the unbound concentrations of the fluoroquinolones, pazufloxacin and ofloxacin, in tissue interstitial fluids (Cisf,u) of rats under steady state conditions. Cisf,u was estimated from the concentration in dialysate and the in-vitro permeability rate constant by the extrapolation method based on the clearance concept. Paper-disks were inserted under the abdominal skin of rats, and the drug concentrations in the fluids penetrating into the disks (Cdisk) were measured and compared with Cisf,u. The Cisf,u of pazufloxacin and ofloxacin in muscle were close to their unbound concentrations in the venous plasma; these were 75.3% and 77.1%, respectively, of the total concentrations in plasma at the steady state. The Cdisk of pazufloxacin and ofloxacin were also close to their Cisf,u. These results indicate that the unbound concentrations of the fluoroquinolones in the tissue interstitial fluids were the same as those in the venous plasma. The disk insertion technique seems to be useful for evaluating drug concentrations in tissue interstitial fluid.

Animals↗
PubMed1997

The predominant contribution of oligopeptide transporter PepT1 to intestinal absorption of beta-lactam antibiotics in the rat small intestine.

Although recent evidence suggests that certain beta-lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of beta-lactam antibiotics was evaluated. For evaluation of transport activity complementary RNA (cRNA) of rat PepT1 was synthesized in-vitro and expressed in Xenopus laevis oocytes. cRNA induced uptake of several beta-lactam antibiotics and the dipeptide [14C]glycylsarcosine; this was specifically inhibited by various dipeptides and tripeptides but not by their constituent amino acids or by tetra- or pentapeptides. The transport activity of PepT1 for beta-lactam antibiotics correlated well with their in-vivo intestinal transport and absorption. Furthermore, mutual inhibitory effects on uptake were observed between glyclsarcosine and beta-lactam antibiotics. Hybrid depletion of the functional expression of rat PepT1 in oocytes injected with rat intestinal epithelial total mRNA was studied using an antisense oligonucleotide corresponding to the 5'-coding region of PepT1. In oocytes injected with rat mRNA pre-hybridized with the antisense oligonucleotide against rat PepT1, the uptake of [14C]glycylsarcosine was almost completely abolished, whereas its uptake was not influenced by a sense oligonucleotide for the same region of PepT1. Similarly, the uptake of beta-lactam antibiotics was also reduced by the antisense oligonucleotide against rat PepT1. These results demonstrate that the intestinal proton-coupled oligopeptide transporter PepT1 plays a predominant role in the carrier-mediated intestinal absorption of beta-lactam antibiotics and native oligopeptides in the rat.

Animals↗
PubMed1997

Intestinal brush-border membrane transport of monocarboxylic acids mediated by proton-coupled transport and anion antiport mechanisms.

Intestinal brush-border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush-border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing-type chambers. [3H]Mevalonic acid uptake by BBMVs showed an overshoot phenomenon in the presence of an inwardly directed proton gradient, but not in the presence of an inwardly directed sodium gradient or an outwardly directed HCO3- or chloride gradient. Initial uptake of mevalonic acid was saturable in the presence of a proton gradient. Uptake of [3H]mevalonic acid was inhibited by various monocarboxylic acids, including acetic acid, benzoic acid, lactic acid, nicotinic acid, pravastatin, salicylic acid and valproic acid, but not by dicarboxylic acid or amino acids. Acetic acid, which is transported by both anion antiport and proton-coupled transport systems, induced serosal bicarbonate-dependent alkalinization in the mucosal-side bathing solution of rabbit jejunal tissues, when examined in Ussing-type chambers. Pravastatin, which is a structural analogue of mevalonic acid and is absorbed via proton-coupled transport like mevalonic acid, did not. The result demonstrates that acetic acid is transported by the bicarbonate-dependent anion antiport system, whereas pravastatin is not. So, it is suggested that monocarboxylic acids are transported by at least two independent transporters, namely, a proton-coupled transporter for most monocarboxylic acids, including mevalonic acid, pravastatin and acetic acid, and an anion antiporter for acetic acid, but not for mevalonic acid or pravastatin. Activation of anion antiporter can induce HCO3- secretion in intact intestine.

Acetic Acid↗
PubMed1997

Decalcification of calcium polycarbophil in rats.

The in vivo decalcification of calcium polycarbophil was examined. The decalcification ratio of [45Ca]calcium polycarbophil in the stomach after oral dosing to rats was more than 70% at each designated time and quite closely followed in the in vitro decalcification curve, indicating that the greater part of the calcium ion is released from calcium polycarbophil under normal gastric acidic conditions. The residual radioactivity in rat gastrointestine was nearly equal to that after oral administration of either [45Ca]calcium chloride + polycarbophil. The serum level of radioactivity was nearly equal to that after oral dosing of [45Ca]calcium lactate. These results indicate that the greater part of orally administered calcium polycarbophil released calcium ions to produce polycarbophil in vivo.

Acrylic Resins↗
PubMed1997

[Chemiluminescent assay for biological substances using lucigenin].

The chemiluminescent reaction of lucigenin with various biological substances has been studied. Chemiluminescence of lucigenin is produced by the addition of either hydrogen peroxide or organic reducing compounds to lucigenin in an alkaline solution. On the basis of these reaction, we have developed highly sensitive chemiluminescent methods for the detection of enzyme immunoassay, especially using alkaline phosphatase as a label enzyme, and also for HPLC of corticosteroids or p-nitrophenacyl esters of carboxylic acids. The detection limits of enzymes were 10(-19)-10(-20) mol per assay, corticosteroids and p-nitrophenacyl esters were 500 fmol per injection.

Acridines↗
PubMed1997

[Chemiluminescent enzyme immunoassay of cholecystokinin in rat plasma using an immuno-affinity column as a pretreatment].

The cholecystokinin (CCK) plasma concentration of a basal level in a normal rat was at a hardly detectable level (a few pg/ml) even using the highly sensitive chemiluminescent enzyme immunoassay. In this paper, we prepared an anti-CCK F(ab')2 fragment which was made by pepsin digestion from the anti-CCK antiserum. The anti-CCK F (ab')2 fragment was immobilized to the Sepharose 4B gel activated BrCN and packed to column. We have extracted and concentrated CCK in the rat plasma using this column and the CCK assayed by the chemiluminescent enzyme immunoassay (CL-EIA) of CCK. The immuno-affinity column extraction system was more accurate and precise than a reversed phase gel extraction system. The mean recovery of CCK from the rat pooled plasma using the immuno-affinity column was 62.2% (n = 12) by the proposed CL-EIA. The mean +/- S. E. (n = 3) of the CCK concentration in the fasted rat plasma is 3.54 +/- 0.10 pg/ml. The plasma CCK concentrations of normal rats and those of camostat administrated rats, could be measured by the proposed CL-EIA using the immuno-affinity column as a pretreatment.

Animals↗
PubMed1997

Values of the serum components in Japanese black beef steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture.

Few reference values for use in metabolic profile tests for the maintenance of high productivity and the prevention of production diseases have been reported in Japanese Black beef cattle. To obtain basic data, 101 healthy steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture, Japan, were examined for the values of their serum components in this preliminary study. At the later fattening stage (5 to 20 months after introduction), statistically significant increases were observed in the mean serum activities of lactic dehydrogenase, glutamic-oxalacetic transaminase, gamma-glutamyl transpeptidase, and creatine phosphokinase, the mean serum contents of triglyceride, total cholesterol, albumin (Alb), total protein, blood urea nitrogen, magnesium, and vitamin E, and the mean serum calcium (Ca)/inorganic phosphorus (IP) ratio, and statistically significant decreases were seen in the mean serum alkaline phosphatase activity and the mean serum contents of glucose, IP, and vitamin A. The mean serum Alb/globulin ratio and the mean serum Ca and nonesterified fatty acids contents demonstrated no statistically significant changes.

Alkaline Phosphatase↗
PubMed1997

[Urinary neopterin levels in patients with genitourinary tract malignancies].

BACKGROUND: Neopterin is released from macrophages upon stimulation with gamma-interferon, secreted by activated T cell. Therefore it has been recognized as a useful indicator of the activation of the T cell-macrophage system. Increased neopterin levels are observed in patients with acute graft rejections, viral infections, auto-immune diseases and several malignancies. Urinary neopterin concentrations were determined in patients with genitourinary tract malignancies to evaluate the usefulness of neopterin as a tumor marker. METHODS: Urinary neopterin concentrations were determined by high-performance liquid chromatography in 90 patients with genitourinary tract malignancies and 28 patients with benign urological tumors and 34 healthy subjects. RESULTS: Increased urinary neopterin levels were observed in 52% of the patients with genitourinary tract malignancies and 7% with benign urological tumors. The positivity rate in patients with renal cell carcinoma (RCC), renal pelvic and ureteral tumor, bladder tumor (BT), prostatic carcinoma (PC) and testicular tumor was 68%, 80%, 47% and 30%, respectively. The difference in the urinary neopterin levels between low and high stages was highly significant (p < 0.0005) in patients with RCC (stage I-II vs. stage III-IV) and BT (T1S-1 VS. T2-4). The urinary neopterin levels were also correlated with the tumor grade in patients with RCC and PC. CONCLUSION: Our study suggests that urinary neopterin levels may supplement laboratory examinations for patients with genitourinary tract malignancies, providing useful information in evaluating the tumor stage and follow-up of the disease.

Adult↗
PubMed1997

Structure-internalization relationship for adsorptive-mediated endocytosis of basic peptides at the blood-brain barrier.

For the purpose of the brain delivery of peptides, the structural specificity of adsorptive-mediated endocytosis at the blood-brain barrier was studied by measuring transport of a newly synthesized basic peptide 001-C8, H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)aNH2, using primary cultured bovine brain capillary endothelial cells. The apparent uptake of [125I]001-C8 increased time-dependently and reached a steady-state at 60 min. The steady-state uptake of [125I]001-C8 was temperature and concentration dependent and was significantly decreased in the presence of dansylcadaverine, protamine or poly-L-lysine. Uptakes of peptides modified by 1,8-octanediamine, 1,5-pentanediamine, 1,2-ethanediamine or ethylamide and peptides with a free carboxyl terminal were significantly higher than, and similar to, that of [3H]PEG900, respectively. The half-saturation constants and the maximal uptake capacities of these peptides were in the ranges of 0.2 to 134 microM and 1.1 to 408 pmol/mg protein, respectively. These values were correlated with the basicity of the molecules. In conclusion, not the number of constituent amino acids of peptides, but rather the C-terminal structure and the basicity of the molecules are the most important determinants for the uptake by the adsorptive-mediated endocytosis system at the blood brain barrier.

Adsorption↗
PubMed1997

[Tissue distribution of the multidrug-resistance gene product P-glycoprotein and its physiological function].

In normal tissues, P-glycoprotein(P-gp), which is expressed in various tumor cells, is found on the luminal surface of epithelia of the kidney proximal tubule, small intestine and colon and bile canalicular face of hepatocytes, as well as, in the adrenal and capillary endothelial cells in the brain and testis. The physiological function of P-gp remains unclear but growing amounts of information suggest that it can play a important role in the absorption from intestine, elimination from liver and kidney and distribution into brain across the blood-brain barrier for many cancer chemotherapeutic agents as well as other drugs which reverse multidrug resistance. The competition for the P-gp-mediated transports in tissues and organs, which express multidrug-transporter, might lead to unsuspected drug interactions among these drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Carrier-mediated hepatic uptake of quinolone antibiotics in the rat.

The systemic clearance of many quinolone antibiotics is mainly via metabolism and urinary excretion; by contrast, biliary excretion is a major route of elimination for a new quinolone grepafloxacin (GPFX). Accordingly, we studied the hepatic uptake of GPFX because it is the first step in the drug's hepatobiliary transport. The hepatic uptake of GPFX in vivo after i.v. administration was found to approach the hepatic blood flow, suggesting the existence of an effective hepatic uptake mechanism. To clarify this transport mechanism, GPFX uptake by isolated rat hepatocytes was examined and found to consist of a saturable component (Km 173 microM, Vmax 6.96 nmol/min/mg) and a nonspecific diffusion component. The inhibition of GPFX uptake by ATP-depletors and a lack of effect after replacing Na+ with choline demonstrated that the uptake was an Na+-independent carrier-mediated active process. This uptake was inhibited by other quinolones and for lomefloxacin this was competitive in nature. Mutual inhibition studies were undertaken to investigate whether the transporter for GPFX might be the same as other transporters so far identified. GPFX inhibited the uptake of taurocholic acid, pravastatin (organic anion), cimetidine (organic cation) and ouabain (neutral steroid). However, GPFX uptake was not inhibited by these compounds. Confirmation that GPFX uptake is blood flow limited was obtained by extrapolation of the in vitro data based on mathematical modeling. In conclusion, the effective hepatic uptake of quinolone antibiotics are via carrier-mediated active transport, which is distinct from that involved in the transport of bile acids, organic anions, organic cations or neutral steroids.

Animals↗