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T Wakamiya

Publications and source records attributed to T Wakamiya.

At least 19 recordsLinked to original sources
PubMed1998

Intestinal absorption of fluorescence-derivatized cationic peptide 001-C8-NBD via adsorptive-mediated transcytosis.

The intestinal absorption of an intact oligopeptide was investigated in rats using a synthetic cationic peptide, 001-C8 (H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2). The peptide was coupled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) to prepare a fluorescence-labeled derivative 001-C8-NBD (H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH-NBD) for the purpose of quantification. The degradation half-life of 001-C8-NBD in jejunal homogenate (1 mg/mL) was 99.5 min, which was significantly longer than that of natural leucine enkephalin (1.14 min). The absorption of 001-C8-NBD was evaluated by the vascular-perfusion method. Intact 001-C8-NBD appeared in the blood time-dependently and the absorption volume at 30 min (2.75 +/- 0.14 microL/cm intestine) was significantly larger than that of [3H]PEG 900 (0.88 +/- 0.13 microL/cm intestine), of which membrane permeability is very low. The absorption of 001-C8-NBD was greatly reduced by an adsorptive-mediated endocytosis inhibitor, protamine (10 mM). No inhibition of the absorption of [3H]PEG 900 by protamine was observed. The intestinal absorption was also measured by an in vivo loop method. The absorption clearance of 001-C8-NBD measured by this method (0.083 +/- 0.008 microL/min/cm intestine) was comparable to that obtained by the vascular perfusion method (0.092 +/- 0.005 microL/min/cm intestine). All of these data suggested that 001-C8-NBD was absorbed as the intact oligopeptide in the intestine in vivo. Adsorptive-mediated transcytosis is suggested to have enormous potential as an oral delivery system for peptide and/or protein drugs.

Animals↗
PubMed1998

Adsorptive-mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells.

The internalization of a basic peptide, 001-C8 [H-MeTyr-Arg-MeArg-D-Leu-NH(CH2)8NH2], into enterocyte-like Caco-2 cells was evaluated. Internalization of 125I-labeled 001-C8 (125I-001-C8) increased time dependently and reached steady state at 60 min. The steady-state internalization of 125I-001-C8 (7.24 +/- 0. 41 microl/mg protein) was temperature and concentration dependent and was significantly decreased by dansylcadaverine (500 microM), protamine (1 mM), poly-L-lysine (1 mM), E-2078 (1 mM), and ebiratide (1 mM), whereas poly-L-glutamic acid (1 mM), tyrosine (1 mM), and glycylglycine (25 mM) were not inhibitory. Predigestion of acid mucopolysaccharides by heparinase I, heparitinase, and chondroitinase ABC also decreased the internalization. The maximal internalization, the half-saturation constant, and the nonsaturable internalization of 125I-001-C8 were 1.13 +/- 0.23 pmol/mg protein, 0. 47 +/- 0.43 microM, and 3.13 +/- 0.19 microl/mg protein, respectively. Confocal microscopy also indicated the internalization of fluorescence-derived 001-C8 [001-C8-4-nitrobenz-2-oxa-1,3-diazole (001-C8-NBD)]. Granular staining seen within the cell, excluding nuclei, indicated the sequestration of 001-C8-NBD within endocytotic vesicles. Dansylcadaverine and protamine strongly decreased the granular distribution of 001-C8-NBD within the cell. These results demonstrate that 001-C8 is taken up by Caco-2 cells via adsorptive-mediated endocytosis.

Caco-2 Cells↗
PubMed1997

Synthetic study of phosphopeptides related to heat shock protein HSP27.

Two kinds of phosphoserine-containing peptides related to HSP27 were synthesized by the Boc- or Fmoc-mode solid-phase method based on prephosphorylation strategy. In the case of the Boc strategy, the O-phosphono group of the phosphoserine residue was protected with the cyclopentyl or cyclohexyl group. On the other hand, N'-Fmoc-O-[(benzyloxy)-hydroxyphosphinyl]serine was employed in case of the Fmoc strategy. Consequently, it has become-feasible to utilize conventional solid-phase methods for synthesizing any phosphopeptides which are required to elucidate biochemical significance of protein phosphorylation.

Chromatography, High Pressure Liquid↗
PubMed1997

Enzyme flexibility: a new concept in recognition of hydrophobic substrates.

The mechanism of recognition of hydrophobic substrates was investigated using Escherichia coli aspartate aminotransferase (AspAT), E. coli aromatic amino acid aminotransferase (AroAT), and their chimeric enzyme (DY18). Surprisingly, broad substrate specificity was observed in the reaction of aminotransferases with hydrophobic substrates. The catalytic efficiency increased with an increase in the side chain length of straight or branched-terminal aliphatic substrates. The straight-chain substrates catalysed with maximal efficiency were the 7-carbon substrate in the case of AspAT and the 8-carbon substrate for AroAT and DY18. Consecutive addition of single methylene groups to the substrate had a constant effect on the stabilization energy of the transition state relative to the unbound state. The dependency of binding energy on each methylene group is usually interpreted as indicating hydrophobicity of the active site. However, we observed that AroAT and DY18 had different dependencies although both enzymes have the same residues in the substrate-binding pocket. For substrates with more than 7 carbons, the aminotransferases did not strictly distinguish between substrates with straight and branched side chains. These results suggest that the recognition of manifold hydrophobic substrates of different shapes might require not only the hydrophobicity of the active site but also enzyme flexibility.

Amino Acid Sequence↗
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↗
PubMed1995

Purification and characterization of [3H]mepyramine (histamine H1 antagonist)-binding protein from rat liver: a highly homologous protein with cytochrome P450 2D.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

Amino Acid Sequence↗
PubMed1990

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain.

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

Amino Acids↗
PubMed1989

Interactive NMR and computer simulation studies of lanthionine-ring structures.

We report progress in elucidating the structure of nisin, a naturally occurring peptide antibiotic. Nisin contains five rings constrained by lanthionine or methyllanthionine bridges, as well as alpha, beta-unsaturated amino acids. We have determined conformations for two model compounds of ring A and a derivative of ring B through interactive nmr and computer simulation studies. High-resolution nmr techniques provides structural information, which was further refined through molecular dynamics simulations. These methods are being applied to the remaining constrained fragments of the molecule. This conformational information will be employed in an aufbau approach to determining the structure of the entire molecule.

Alanine↗
PubMed1989

Lanthiopeptin, a new peptide antibiotic. Production, isolation and properties of lanthiopeptin.

A strain of Streptoverticillium cinnamoneum produced a peptide antibiotic named lanthiopeptin, which contained four unusual amino acids, erythro-beta-hydroxyaspartic acid, mesolanthionine, threo-beta-methyllanthionine and lysinoalanine. Lanthiopeptin showed antiviral activity against herpes simplex virus type 1 KOS strain infection in Vero cells by cytopathic effect reduction assay. The structure of lanthiopeptin is similar to that of ancovenin.

Amino Acid Sequence↗
PubMed1985

N tau-Ribosylhistidine, a novel histidine derivative in urine of histidinemic patients. Isolation, structure, and tissue level.

On amino acid analysis of urine of histidinemic patients, an unidentified compound was eluted in a position between beta-aminoisobutyric acid and gamma-aminobutyric acid. This compound was purified to homogeneity from the urine by a combination of extraction with 80% ethanol, repeated column chromatography on Bio-Rad AG-50, and high performance liquid chromatography on a strongly cationic ion exchanger. The compound yielded free histidine on hydrolysis in an evacuated sealed tube with 0.1-6.0 M HCl at 145 degrees C for 5 h, but not at 100 degrees C for 24 h. This compound was determined to be N tau-ribosylhistidine by 1H and 13C NMR spectroscopies. The urinary content of this material in normal and histidinemic children was 17.8 +/- 13.4 (n = 10) and 126 +/- 51 (n = 14) mumol/g creatinine (mean +/- S.D.), respectively, and were closely correlated with those of urinary histidine. The renal clearance value of N tau-ribosylhistidine in humans was 96% of that of creatinine. When rats were fed on diets rich in histidine, the urinary excretion of N tau-ribosylhistidine increased greatly and was well correlated with the intake of histidine.

Amino Acid Metabolism, Inborn Errors↗
PubMed1984

Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by Actinomadura sp.

A new inhibitor of angiotensin I converting enzyme, I5B2, was isolated from the culture broth of Actinomadura sp. No. 937ZE-1. This compound contains N-methylvaline, tyrosine and 1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid. The microorganism also produced another inhibitor, I5B1, which is identical with K-4 isolated from Actinomadura sp. as an antihypertensive agent.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Antibacterial activity of palmitoyltuberactinamine N and di-beta-lysylcapreomycin IIA.

Palmitoyltuberactinamine N (Pal-Tua N) and di-beta-lysylcapreomycin IIA (di-beta-Lys-Cpm IIA), which are synthetic derivatives of the antituberculous agent tuberactinomycin (Tum) and capreomycin (Cpm) respectively, were tested for anti-bacterial activity. Pal-Tua N inhibited not only tuberactinomycin-resistant Mycobacterium smegmatis but also Escherichia coli, Corynebacterium diphtheriae, Staphylococcus aureus, Streptococcus pyogenes, although it has lost activity against Mycobacterium tuberculosis. Di-beta-Lys-Cpm IIA inhibited the growth of laboratory-derived Tum-resistant M. smegmatis and M. tuberculosis as well as Tum-resistant M. tuberculosis from patients with one exceptional case.

Anti-Bacterial Agents↗
PubMed1979

Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVPS/1/P2 (EcoRI res).

The entire nucleotide sequence (1210-base-pair repeating units) of a defective variant of simian virus 40 is presented. Within this variant there are deletions of large portions of the wild type genome and an inversion within the remaining wild type viral sequences. In addition, the defective variant contains DNA sequences derived from the permissive monkey cells in which the virus was propagated. The monkey sequences include a portion that is homologous to sequences within highly repeated monkey DNA (alpha component) as well as portions derived from sequences that are infrequently repeated in the monkey genome. One out of every three to four of the tandem 1210-base-pair repeat units contains in addition, a duplication of a part of the monkey sequences. The sequence information defines the structures of a number of recombinational joints which result from deletions, inversions, duplications, and insertions of host sequences into the viral genome. The data demonstrate that the various recombinational events that resulted in the formation of this defective variant did not depend on extensive homology between recombining segments.

Animals↗
PubMed1977

Chemical studies on tuberactinomycin. XV. Total synthesis of tuberactinomycin O.

Tuberactinomycin O, one of the four congeners of the antituberculous peptide tuberactinomycin, was totally synthesized. The beta-ureidodehydroalanine moiety was constructed from beta,beta-diethoxyalanine with excess urea in acidic medium after a cyclization reaction of a pentapeptide was finished. Cyclization was carried out by means of the 1-succinimidyl ester method. To the cyclic pentapeptide, beta-lysine was introduced as the branched moiety and then deprotected to afford tuberactinomycin O which was completely identified with the natural form of the antibiotic.

Antibiotics, Antitubercular↗
PubMed1977

The revised structure of capreomycin.

The total structures of capreomycins IA and IB were determined mainly from the results of the comparative study on the NMR spectra of capreomycins and tuberactinomycins, resulting in a revision of the formerly proposed structure. The beta-lysine residue as a branched part was revealed to be linked with the beta-amino group of the alpha, beta-diaminopropionic acid residue in the cyclic pentapeptide moiety in a different manner than in tuberactinomycins.

Amino Acid Sequence↗