Effect of disulfiram on turnover of 5-hydroxytryptamine in rat brain.

Effect of disulfiram on 5-hydroxytryptamine (5-HT) turnover was studied. Treatment with disulfiram caused increases in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in rat brain. Under the same condition, activity of brain mitochondrial aldehyde dehydrogenase was reduced, however, supernatant aldehyde dehydrogenase and monoamine oxidase activities remained unchanged. Disulfiram had no effect on synthesis rate of 5-HT, but decreased metabolism of 5-HT. Moreover, disulfiram impaired transport of 5-HIAA from brain tissue.     

A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.     

Elevation of nucleotide pyrophosphatase activity in skin fibroblasts from patients with Lowe's syndrome

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient[Fukui, S. etal. (1981) J. Biol. Chem. 256, 10313–10318] was found to be caused by elevated degradation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The enzyme involved in this degradation was then identified as an enzyme of nucleotide pyrophosphatase (EC 3.6.1.9) nature, cleaving the phosphosulfate linkage. The specific activities were 8 – 24 (mU/mg protein) in patients' fibroblasts, in contrast to 3 in normal and 5 – 14 in heterozygote cells. A possibility is discussed that the elevation of nucleotide pyrophosphatase activity is the primary genetic defect in Lowe's syndrome.     

Effects of anions on ADP-stimulated aggregation of bovine blood platelets

ADP-stimulated aggregation of bovine blood platelets was observed in media containing isotonic potassium salts of various monovalent anions. The aggregation depended on the anion in the medium, the order of aggregation being Cl^?, Br^?>I^?>SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$. After 30-min incubation, the extent of aggregation of platelets in Cl^? or Br^? medium was little changed, whereas, that in SCN^? or $\text{ClO}{}_4{}^{\mathrm{?}}$ medium was remarkably decreased. This anion dependency of aggregation may be due to change in the membrane potential.     

The effect of modifications of the A5 and A19 amino acid residues on the biological activity of insulin. [Leu5-A] and [Phe19-A] sheep insulins

Two analogs of sheep insulin, both differing from the native material by a single amino acid in the A chain, have been synthesized and isolated in highly purified form by procedures developed in this laboratory. In one case, the glutamine residue in position A⁵ was replaced by leucine ([Leu⁵-A]); in the other, the tyrosine residue in position A¹⁹ was replaced by phenylalanine ([Phe¹⁹-A]). The biological behavior of these analogs was compared with natural bovine insulin inin vitro tests and in receptor-binding assays, as well as in radioimmunoassay. In the stimulation of glucose oxidation by rat adipocytes, the analogs gave relative potencies of 30% and 7.8% for [Leu⁵-A] and [Phe¹⁹-A], respectively. Receptor-binding assays in rat liver plasma membranes showed similar behavior for both analogs. In radioimmunoassay, [Leu⁵-A] displayed a relative potency of 27.9%, while [Phe¹⁹-A] showed a relative potency of 19–27%, compared with bovine insulin. At high concentration, both analogs displayed the same maximal activity as bovine insulin, and the dose-response curves are essentially parallel. It is speculated that the interaction between the glutamine residue in position 5 and the tyrosine residue in position 19 of the A chain of insulin are important in maintaining a three-dimensional structure commensurate with high biological activity. The full intrinsic activity of both analogs at high concentrations and the similarity of the potency figures in receptor-binding and glucose-oxidation assays permit the further conclusion that the reduced potency in the latter assay can be ascribed wholly to the reduced binding affinity toward insulin receptors caused by the substitutions made in the analogs. The receptor-analog complexes are fully capable of triggering the next event in the chain leading to the biological response.     

N-alpha-carbobenzoxy pyroglutamyl diazomethyl ketone as active-site-directed inhibitor for pyroglutamyl peptidase

Pyroglutamyl-peptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.19.3) from Bacillus amyloliquefaciens was covalently labeled with a newly synthesized N-carbobenzoxy-L-pyroglutamyl diazomethyl ketone (Z-PGDK) and was completely inactivated. The inactivation reaction proceeded in pseudo-first order. The kinetic studies demonstrated a rate-limiting step in the inhibition reaction, resulting in the formation of a reversible (enzyme.reagent) complex. The calculated KI,app is 0.12 mM at pH 7.58. The rate of inactivation was pH dependent with an extrapolated pK value of approx. 8.6. The enzyme could be protected against inactivation by a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that could be reversibly reactivated by mercaptoethanol, failed to react with Z-PGDK. The enzyme was insensitive toward the D-isomer of Z-PGDK and other diazomethyl ketone derivatives of carbobenzoxy amino acids such as Z-L-proline and Z-L-phenylalanine. These results strongly suggest that the Z-PGDK reacts as an affinity label, presumably with a cysteine residue as the site of alkylation in pyroglutamyl-peptidase, as was reported for chloromethyl ketone derivatives of pyroglutamic acid and its N-carbobenzoxy derivative.     

Isolation and characterization of the 160,000-Da phosphotyrosyl protein, a putative participant in insulin signaling.

The 160,000-Da protein (pp 160) which is rapidly phosphorylated on tyrosine in response to insulin and thus is a putative participant in signaling from the insulin receptor has been purified to homogeneity from 3T3-L1 adipocytes. Isolation of this protein was accomplished by chromatography on an immobilized monoclonal antibody against phosphotyrosine, followed by gel electrophoresis. Sufficient protein was obtained to allow the determination of the sequences of several peptides, which in turn enabled the development of anti-peptide antibodies that specifically recognize pp 160. Immunoblotting of 3T3-L1 adipocyte lysates, together with the purified pp 160 as a standard, indicate that an insulin-treated 3T3-L1 adipocyte possesses about 230,000 copies of tyrosine-phosphorylated pp160 and that this amount is approximately 25% of the total pp160 in the cell. The number of tyrosine-phosphorylated pp160s per cell is approximately the same as that of insulin receptor beta subunits. These results provide further evidence for a role of pp160 in insulin signaling. Moreover, the availability of purified protein and knowledge of peptide sequences will allow the elucidation of the structure and function of this protein.     

Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.