ABSTRACT
After mutagenesis with nitrosoguanidine and selection by immuno-halo techniques, an avirulent mutant, designated Texas Star-SR, which produces no detectable A (active; ADP-ribosylating) region of the cholera enterotoxin (choleragen) but produces the B region (choleragenoid) in amounts similar to the hypertoxinogenic wild-type parent Vibrio cholerae (biotype E1 Tor serotype Ogawa), has been isolated. The mutant retains the colonizing ability, motility, prototrophy, and serologic characteristics of the parent. In relevant intestinal experimental models, it has been shown to be avirulent and to induce protection against challenge with virulent cholera vibrios. The mutant appears to be suitable for further evaluation in volunteers as a candidate living enteric vaccine against cholera and related enterotoxic enteropathies.
Subject(s)
Adenosine Diphosphate Sugars/analysis , Cholera Toxin/analysis , Nucleoside Diphosphate Sugars/analysis , Vibrio cholerae/isolation & purification , Animals , Biological Assay , Cholera Toxin/pharmacology , Ileum/drug effects , Immunodiffusion , Mutation , Rabbits , Ribose , Species Specificity , Vibrio cholerae/geneticsABSTRACT
A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5'-AMP by alkaline treatment. Antibodies highly specific against 5'-AMP allowed quantification of ADP-ribose converted to 5'-AMP in the range of 1-40 pmol, and in the presence of large quantities of nucleic acids or 3'-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5'-AMP from the non-reducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5'-AMP and of the radioimmunoassay for 5'-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.
Subject(s)
Adenosine Diphosphate Sugars/analysis , Nucleoside Diphosphate Sugars/analysis , Poly Adenosine Diphosphate Ribose/analysis , Animals , Animals, Newborn , Carcinoma, Ehrlich Tumor/analysis , Cross Reactions , Liver/analysis , Mice , Molecular Weight , Radioimmunoassay/methods , Rats , RiboseABSTRACT
ADP-Ribose is nearly quantitatively split to 5'-AMP by treatment with alkali at elevated temperatures. This unique behaviour, which is not shown by ADP and other adenine derivatives, was used as the basis of an optical test for the selective determination of ADPR in the presence of other adenine compounds including RNA. Poly(ADPR) could also be quantified when the polymer was degraded by poly(ADPR) glycohydrolase prior to alkaline treatment. When combined with the determination of the terminal AMP residues released by phosphodiesterase I treatment, the chain length of the polymer could be calculated. Application of the method to the quantitation of protein-bound mono(ADPR) residues in Ehrlich ascites tumor cells under different growth conditions is described.
Subject(s)
Adenosine Diphosphate Sugars/analysis , Nucleoside Diphosphate Sugars/analysis , Poly Adenosine Diphosphate Ribose/analysis , Adenosine Monophosphate , Animals , Carcinoma, Ehrlich Tumor/analysis , Microchemistry/methods , RiboseABSTRACT
Evidence is presented for the structural differences between rat and rabbit skeletal muscle glyceraldehyde-3-phosphate dehydrogenases. Glyceral-dehyde-3-phosphate dehydrogenases isolated from frozen and fresh rat skeletal muscle were found to have different temperature stability and chromatographic behavior on CM-cellulose. Different nucleotide content of the preparations (NAD on one hand, and products of its degradation - ADP-ribose, AMP and adenosine, on the other) is shown to account for the observed effects.
