Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.     

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.     

Somatostatin inhibits neutrophil elastase release in vitro

The influence of the long-acting somatostatin analogue, SMS 201-995, on FMLP-induced neutrophil elastase release in vitro has been investigated. Doses from 150 ng/ml upwards inhibited elastase release, with 100% inhibition by 2500 ng/ml. Inhibition was demonstrated both by an assay measuring elastase immunometrically and by an assay based on its enzyme activity. The demonstration that SMS 201-995 inhibits protease release from polymorphonuclear leukocytes may have implications for the long-term clinical use of this somatostatin analogue.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and -naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied. (Mol Cell Biochem120: 171–179, 1993)Thesis student of the Graduate School of Biomedical Sciences, the University of Texas Health Science Center at Houston     

Effects of Interactions BetweenPasteurella haemolyticaandBordetella parapertussisonin vitroPhagocytosis by Lung Macrophages

The aim of the work was to determine the effect of exposing ovine bronchoalveolar macrophages (BAM)in vivotoPasteurella haemolyticaand/orBordetella parapertussison the subsequent uptake and killing ofP. haemolyticaby these cellsin vitro. Exposurein vivotoP. haemolyticadid not affect the uptake ofP. haemolyticaby BAMin vitrobut reduced (P< 0·05) the intracellular killing of bacteria. Exposurein vivotoB. parapertussishad no significant effect on either the uptake of killing ofP. haemolytica in vitro. However, sequential exposurein vivotoB. parapertussisandP. haemolyticareduced both the ingestion (P< 0·05) and killing (P< 0·001) ofP. haemolytica in vitro. These results indicate that exposure toP. haemolyticacompromised the bacterial killing mechanisms of BAM and that synergy betweenB. parapertussisandP. haemolyticareduced the ability of BAM to ingest bacteria.     

Sexual development of patients with isochromosomes for the long arm of the X chromosome

Summary The sexual development of 14 girls with non-mosaic monocentric 46,X,iXq karyotype was studied. Seven out of eight girls were found to have immature secondary sexual characteristics and amenorrhoea, a finding greatly contrasting with that in Triplo-X girls. The relative ineffectiveness of the isochromosome Xq in maintaining fertility may be due to the absence of one short arm, which probably also carries a gonadal determinant. Alternatively, the presence of two inactivation sites on one isochromosome may render the gonadal determinants inactive at an important stage in gonadal development.     

Helical channels in crystals of gramicidin A and of a cesium--gramicidin A complex: an x-ray diffraction study.

X-ray crystallographic studies of gramicidin A crystallized from methanol (P2₁) and ethanol (P2₁2₁2₁), and of a Cs⁺ gramicidin A complex crystallized from methanol (P222₁, P2₁2₁2 or P2₁2₁2₁) are reported here. The asymmetric unit consists of two molecules of gramicidin A in the native crystals and four molecules in the cesium complex crystal. Patterson analyses show that gramicidin A in these crystals forms a cylindrical helical channel. In the two types of native gramicidin crystals, the diameter of this channel is about 5 å and its length is about 32 å. Cesium ions are bound inside this channel in crystals of the cesium-gramicidin A complex. The channel in this complex is considerably shorter (26 Å) and wider (6·8Å) than in the native forms. The Patterson maps of these three crystal forms are compatible with either the single-stranded β-helix (Urry, 1971) or the double-stranded parallel or anti-parallel, β-helix (Veatch et al., 1974).