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D R Webster

Publications and source records attributed to D R Webster.

At least 37 records · Page 2Linked to original sources
PubMed1987

Differential turnover of tyrosinated and detyrosinated microtubules.

Turnover of tyrosinated and detyrosinated microtubules ([Tyr]MTs and [Glu]MTs, respectively) was analyzed by the combined use of hapten-mediated immunocytochemistry and peptide-specific antibodies. Cells were microinjected with hapten-labeled tubulin and then processed for triple-label immunofluorescence to determine the pattern of incorporation of the injected subunits into [Tyr]- and [Glu]-MTs. Within 2 min of microinjection, hapten-labeled domains were present at the ends of virtually all [Tyr]MTs but were absent from most [Glu]MTs, demonstrating that [Tyr]MTs grew, whereas most [Glu]MTs did not. After 1 hr of incubation, all [Tyr]MTs analyzed were copolymers of endogenous and hapten-labeled subunits, indicating complete and rapid turnover of these MTs. However, the majority of [Glu]MTs were not hapten-labeled, indicating that they had not turned over. Even 16 hr after injection, cells that had not divided retained a small proportion of [Glu]MTs lacking hapten, implying that some had persisted for most of a cell generation. At mitosis, all MTs were hapten-labeled, indicating that the stable interphase [Glu]MTs had depolymerized. The results establish that the MT network is heterogeneous in its turnover rate, being composed of at least two populations: [Tyr]MTs that turn over rapidly and [Glu]MTs that turn over slowly.

Animals↗
PubMed1987

Assembly and turnover of detyrosinated tubulin in vivo.

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.

Animals↗
PubMed1986

Relationship of metabolism of 2'-, 3'- and 5'-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens.

In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2'-, 3'-, and 5'-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole. The metabolism of these nucleotides was examined utilizing HPLC analysis of the bathing medium after exposure to 30 microM nucleoside or nucleotide for 5 min. 5'-AMP, 5'-ADP, 5'-ATP, and NAD+ were all partially hydrolysed to adenosine, the relative extent of this being 5'-AMP greater than 5'-ADP = 5'-ATP greater than NAD+. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products. These results indicate that the 2'-, 3'- and 5'-substituted nucleotides studied act at a P1-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5'-AMP, 5'-ADP, 5'-ATP and NAD+, all appear to act directly at this receptor. However, the 5'-adenine nucleotides (AMP, ADP and ATP) and NAD+ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.

Adenine Nucleotides↗
PubMed1985

Measurement of adenosine metabolites and metabolism in isolated tissue preparations.

Several pitfalls have been encountered in setting up methodology for the study of adenosine metabolism and quantitation of adenosine metabolites in isolated tissue preparations. Quantitation of metabolites by high-performance liquid chromatography (HPLC) methods has shown that: 1. significant absorbtion of purines to some types of thread (used to tie up tissues in tissue baths) occurs; 2. without particular care in the preparation of tissue baths and medium, significant microbiological contamination can occur; 3. stimulation of guinea pig ileum preparations at 0.2 Hz is associated with weight loss of the tissue and release of purine-degrading enzymes into the bathing medium; 4. stability of purine analogs is dependent on the tissue and species. A method for the measurement of the purines uric acid, xanthine, hypoxanthine, inosine, adenosine, adenine nucleotides, and various substituted nucleotides in medium and tissue samples following incubations of rat vas deferens and guinea pig ileum is presented.

Adenosine↗
PubMed1985

An inborn error of purine metabolism, deafness and neurodevelopmental abnormality.

A syndrome of hyperuricemia, sensorineural deafness, mild mental handicap and congenital disequilibrium in a four-year-old boy is probably inherited as a sex-linked condition since his mother has sensorineural deafness and similar biochemical abnormalities. There is evidence of a superactive PP-ribose-P synthetase, normal purine salvage enzymes, and severe depletion of nicotinamide adenine dinucleotide and guanine triphosphate in red cells.

Deafness↗
PubMed1985

Mechanism of presynaptic inhibition of cholinergic transmission in guinea-pig ileum by adenine nucleotides.

This study was designed to determine the mechanism of action of 2'-, 3'- and 5'-substituted adenine nucleotides on cholinergic transmission in guinea-pig ileum. Segments of ileum were continuously stimulated at 0.2 Hz and IC50 values of nucleotides for inhibition of twitch responses determined. All the nucleotides studied inhibited transmission, the effect being antagonised by 30 X 10(-6) M theophylline. The inhibitors of nucleoside transport, HNBTGR and dipyridamole, potentiated responses to all the nucleotides. Addition of adenosine deaminase reduced responses only to adenosine and 5'-AMP, while inhibition of adenosine deaminase with deoxycoformycin potentiated responses only to 5'-AMP and 5'-ADP. It was concluded that all the nucleotides studied inhibit cholinergic transmission through an action at P1-purinoceptors. However, it was not possible from this study to conclude whether these actions were direct or indirect following their hydrolysis to adenosine.

Adenine Nucleotides↗
PubMed1985

EHNA is a poor inhibitor of deoxyadenosine catabolism in cultured human lymphocytes.

Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) has been used by many workers as enzyme inhibitor in vitro to simulate the in vivo situation in inherited adenosine deaminase (ADA) deficiency. In this study the metabolism of 8-14C deoxyadenosine (dAR) has been followed in cultured lymphocytes from patients deficient in enzymes associated with the catabolism and salvage of dAR, in the absence and presence of 10 microM EHNA. The results show that EHNA, at these concentrations, does not prevent the catabolism of dAR and thus does not provide a valid model for investigating the toxicity to the immune system in inherited ADA deficiency.

Adenine↗