Biomedical subjects

T Agui

Publications and source records attributed to T Agui.

62 records · Page 4Linked to original sources

PubMed1988

Binding of [125I]-N-(p-aminophenethyl)spiroperidol to the D-2 dopamine receptor in the neurointermediate lobe of the rat pituitary gland: a thermodynamic study.

The novel iodinated ligand [125I]-N-(p-aminophenethyl)spiroperidol ([125I]NAPS) was used to identify the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland. The binding of [125I]NAPS was of high affinity and saturable, given that the dissociation constant and the maximal binding were 34.7 +/- 4.8 pM and 21.1 +/- 2.5 fmol/mg of protein, respectively. The ability of dopaminergic agonists and antagonists to compete with [125I]NAPS varied markedly with incubation temperature. The marked decrease of the molar potency associated with increasing incubation temperature in the competitive displacement curve suggested that the binding of five agonists, dopamine, (-)-apomorphine, (-)-n-propylnorapomorphine, N-0434, and LY-171555, to the D-2 dopamine receptor was enthalpy-driven, with a negative change in entropy. In contrast, the binding of three antagonists, fluphenazine, (+)-butaclamol, and domperidone, was entropy-driven, with positive change in entropy, suggesting less temperature-sensitive change in the molar potency. Several molecules gave unanticipated results; the molar potency of two dopamine agonists, bromocriptine and lisuride, was much less temperature-sensitive than the other agonists used in this study. The thermodynamic parameters for the atypical agonists indicated entropy-driven binding. Conversely, the molar potency of (+)-apomorphine, a dopamine receptor antagonist, was markedly affected by incubation temperature, indicating enthalpy-driven binding. Another antagonist, YM-09151-2, was affected by the inclusion of sodium chloride in the assay system: in the absence of sodium chloride, the drug was relatively weak and displayed enthalpy-driven binding; in the presence of sodium chloride, its molar potency was increased and its binding manner turned into entropy-driven.

Animals↗

PubMed1987

125I-iodinated benzazepines bind to melanin: implications for the noninvasive localization of pigmented melanomas.

Both the 5-R and the 5-S enantiomers of [125I]2,3,4,5-tetrahydro-8-iodo-3-methyl-5-phenyl-1H-3-benzazepin- 7-ol bind to melanin. The interaction between the 5-R enantiomer and melanin permits visualization of melanomas in mice with a noninvasive imaging procedure. Two lines of evidence suggest that the interaction between iodinated ligands and melanin is not related to the D-1 dopamine receptor, a known target for the 5-R enantiomer: first, melanin binds both enantiomers of the 125I-iodinated benzazepine while the D-1 receptor binds only the 5-R enantiomer; second, the melanin binding site displays only a 5-fold difference in affinity towards the R- and S-enantiomers of SCH 23390 while the D-1 receptor displays a 100-fold difference in affinity towards these two molecules. Because both enantiomers of the iodinated benzazepine bind to a human pigmented melanoma, we propose that such compounds may be of use in the diagnosis of pigmented melanoma: in addition, we discuss the possible application of these molecules as a supplement to existing technology for the localization of pigmented melanomas.

Animals↗

PubMed1986

Detection of a serum class I molecule in rat with anti-rat liver beta-2-microglobulin.

Rat serum was fractionated on a column of Sephacryl-300 and tested with a rabbit anti-rat beta-2-microglobulin (B2m) antiserum. This antiserum was directed against B2m purified from rat liver, and its specificity was confirmed by immunoprecipitation procedures. The antiserum recognized three peaks in the fractionated rat serum: a 200- to 300-kd (kilodalton) fraction, a 40- to 70-kd component, and the free 12-kd B2m. Indirect immunoprecipitation from the 200- to 300-kd fraction led to the identification of a 43-kd polypeptide associated with B2m. A xenoantiserum against RT1 class I antigen also precipitated a similar polypeptide from the same fraction, but this molecule differed in size and antigenic specificity from the one precipitated by anti-rat B2m.

Animals↗

PubMed1985

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin.

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Animals↗

PubMed1983

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes.

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.

Animals↗

PubMed1982

Four different monoclonal antibodies against type C1 toxin of Clostridium botulinum.

Monoclonal antibodies against type C1 toxin produced by Clostridium botulinum type C strain Stockholm (C-ST) were prepared by fusion of BALB/c myeloma cells P3X63-Ag8, with spleen cells from the mice immunized by C-ST toxoid. About 5% of single-cell colonies in wells were found to produce antibodies against the toxin as determined by an enzyme-linked immunosorbent assay (ELISA). Four different hybridoma cell lines, no. 9, 12, 14, and 17, were established, cloned by limiting dilution, and intraperitoneally injected into mice to obtain the ascites fluids containing high-titered antibodies. The reactions of these antibodies to type C1 and D toxins of strains C-ST, D-1873, and D-South African (D-SA) were observed by both neutralization and ELISA tests. Three monoclonal antibodies, no. 9, 14, and 17, reacted with C-ST toxin, but only no. 17 highly neutralized the toxin. These antibodies did not react with type D toxins. On the contrary, no. 12 reacted with toxins of both C-ST and D-SA (but not of D-1873) and commonly neutralized these two toxins. This indicates that there is a common antigenic part between C-ST and D-SA toxin molecules which participates in the toxin-neutralizing reaction. The neutralization profiles of C-ST toxin by no. 12 and 17 antibodies were different in a time-to-death test of mice. The mechanisms of neutralization by no. 12 and 17 may be different.

Animals↗

PubMed1981

Homogeneity and heterogeneity of toxins produced by Clostridium botulinum type C and D strains.

Five Clostridium botulinum strains were used in the present work, two of type C, C-Stockholm (C-ST) and C-CB19, and three of type D, D-South African (D-SA), D-1873, and D-CB16. The toxins, except for those of C-CB19 and D-CB16, were purified, and antisera were prepared in rabbits. To clarify the antigenicity of the toxins, neutralization and agar gel double-diffusion tests were performed. Anti-C-ST toxin serum neutralized two kinds of type C(1) toxin to a similar extent. Antisera against D-SA and D-1873 toxins, however, showed different neutralizing activity toward three type D toxins. Precipitin lines formed between D-SA and D-1873 toxins, and their antisera spurred to each other. From anti-D-SA toxin serum, two neutralizing fractions, one which neutralized D-SA, D-1873, and D-CB16 and one which neutralized only D-SA, were obtained. These results indicate that the antigenicities of D-SA and D-1873 toxins are not identical. Anti-C-ST toxin serum produced cross-neutralization on type D toxins, although the neutralization titer differed depending on the kind of toxin used. A precipitin line was formed with D-SA toxin, but not with D-1873; the developed line spurred to the C-ST toxin precipitin line. Two anti-D toxin sera also caused cross-neutralization on type C(1) toxins. However, the neutralizing activity of each serum to the same type C(1) toxin was different, and only anti-D-SA toxin serum developed a precipitin line with C-ST toxin which spurred to the D-SA toxin precipitin line. From anti-D-SA toxin serum, two different fractions capable of neutralizing C(1) and D toxins were obtained; one neutralized C-ST, C-CB19, and D-SA toxins, but not D-1873 and D-CB16, and the other neutralized all five toxins. There may be at least two common parts among C-ST, C-CB16, and D-SA toxin molecules.

Antigens, Bacterial↗

PubMedDate not supplied

Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland.

The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [125I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [125I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [125I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland.

Animals↗