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.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Class I and class II restriction pattern polymorphisms associated with independently derived RT1 haplotypes in inbred rats

This communication reports the DNA level identification of class I and class II sequences associated with 20 RT1 haplotypes which have been assigned previously to eight RT1 groups. Sixteen to 22 bands in genomic blots hybridized with the mouse pH-2III class I cDNA probe. Only the three RT1 ^khaplotypes associated with identical class I restriction fragment patterns. Differences in restriction bands between putatively identical RT1 haplotypes were either less than or equal to 6%, or greater than 50%, suggesting a relatively high level of recombination between serologically identified RT1.A genes and the majority of class I sequences. Restriction fragment patterns associated with three RT1 ^uhaplotypes differed by less than 6%. However, intra-RT1 ^a,intra-RT1 ^b,and intra-RT1 ^lrestriction fragment differences were between 50 and 64%. In specific cases, different RT1 haplotypes associated with identical class I restriction patterns, e.g., RT1 ^m(MNR) and RT1 ^d(MR); higher resolution confirmed the difference (two bands) between RT1 ^mand RT1 ^d.Results of hybridization with the human DC₁ probe confirmed that the AVN RT1 ^aand NSD RT1 ^bhaplotypes were generated by recombinations within the vicinity of the RT1.B : RT1.D regions. These results demonstrate that a previous classification of RT1 haplotypes was incomplete and did not include the majority of class I and class II sequences which distinguish RT1 haplotypes.     

Evidence for the importance of histidine at the active site of argininosuccinate synthetase from soybean

Studies to determine the role of histidine in catalysis by L-argininosuccinate synthetase (EC 6. 3. 4. 5) were carried out with the enzyme isolated from soybean cell suspension cultures. These experiments utilized analogues of the substrates citrulline and aspartate to investigate substrate binding, and to determine which portion of the molecule were required for binding at the active site of the enzyme. Photooxidation studies using rose bengal were carried out to define the importance of histidine residues for catalysis. These studies suggest that an active site histidine residue has an important role to play in the formation of argininosuccinate by this enzyme.     

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Weight differences, brood reduction, and sibling competition among nestling Stonechats Saxicola torquata (Aves: Turdidae)

Weight differences within broods of nestling birds are commonly assumed to be the outcome of a parental strategy of selectively starving one or more offspring so as to enhance the survival of the remaining chicks. This study examined patterns of development of nestling Stonechats to reveal the significance of weight hierarchies in determining the relative growth and survival of siblings. Although Stonechat broods display obvious weight hierarchies, the results show that a low rank does not strongly influence survival within the nest, especially early in the nestling period, and in small broods. Low ranking chicks were more likely to leave the nest at lighter weights, however. A proximate mechanism for the establishment of weight hierarchies was investigated, involving competition among nestlings for advantageous positions in the nest, in which the heavier siblings were more successful.