Search PubMed⌕ Search

Biomedical subjects

D A Wright

Publications and source records attributed to D A Wright.

At least 73 records · Page 4Linked to original sources
PubMed1989

Effects of 4 h preexercise carbohydrate feedings on cycling performance.

This study determined the effects of consuming three different amounts of liquid carbohydrate 4 h before exercise on the metabolic responses during exercise and on exercise performance. Four hours before exercise subjects consumed either 45 (L) or 156 (M) g of carbohydrate in isocaloric feedings and either 0 (P) or 312 (H) g of carbohydrate. Interval cycling was undertaken for 95 min, followed by a performance trial. Blood glucose had reached basal 1 h after all feedings; blood insulin had reached basal 3 h after ingestion of P, L, and M but was still 84% higher for H at the start of exercise. During exercise insulin averaged 48% higher for H than P. Blood glucose decreased 16% during exercise for P, L, and M, whereas for H there was a transient drop the first 15 min of exercise, after which glucose increased and remained constant throughout exercise. More carbohydrate oxidation occurred during exercise for H vs P, whereas results were similar for L and M. Ingestion of H improved performance by 15% as compared with P, whereas performance was similar for L and M. These results indicate that, despite elevated insulin at the start of and during exercise, consumption of 312 g of carbohydrate 4 h before moderately intense prolonged exercise can improve performance, perhaps via an enhancement of carbohydrate oxidation.

Adult↗
PubMed1988

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.

Animals↗
PubMed1988

Four phosphoproteins with common amino termini are encoded by human cytomegalovirus AD169.

In this report, we identify the proteins encoded by the 2.2-kilobase class of early transcripts arising from a region of the strain AD169 human cytomegalovirus genome (map units 0.682 to 0.713) which contains cell-related sequences. These transcripts, encoded by adjacent EcoRI fragments R and d, have a complex spliced structure with 5' and 3' coterminal ends. Antiserum directed against a synthetic 11-amino-acid peptide corresponding to the predicted amino terminus of the proteins was generated and found to immunoprecipitate four infected-cell proteins of 84, 50, 43, and 34 kilodaltons. These proteins were phosphorylated and were associated predominantly with the nuclei of infected cells. The 43-kilodalton protein was the most abundant of the four proteins, and its level of expression remained relatively constant throughout the infection. Expression of the other proteins increased as the infection progressed. Pulse-chase analysis failed to show a precursor-product relationship between any of the proteins. A comparison of the [35S]methionine-labeled tryptic peptide maps of the four proteins from infected cells and an in vitro-generated polypeptide derived from the putative first exon showed that all four infected-cell proteins were of viral origin and contained a common amino-terminal region.

Amino Acid Sequence↗
PubMed1987

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.

Animals↗
PubMed1986

Isolation of a genomic clone for Drosophila sn-glycerol-3-phosphate dehydrogenase using synthetic oligonucleotides.

A genomic clone containing Drosophila sn-glycerol-3-phosphate dehydrogenase sequences has been isolated using a mixture of synthetic tridecanucleotides as a hybridization probe. The clone as well as the synthetic probe mixture was found to hybridize to an abundant poly(A)+ RNA of 1700 bases. A partial DNA sequence obtained for a 40-amino acid region containing the oligonucleotide hybridization site was found to agree with the known Drosophila protein sequence data for this region of the protein. In situ hybridization of this clone to the polytene chromosomes of wild type flies and flies bearing chromosomal aberrations that delimit the Gpdh+ locus have allowed us to decisively place the gene in the distal region of 26A on the left arm of the second chromosome.

Amino Acid Sequence↗
PubMed1986

Autosomal modifiers of the bobbed phenotype are a major component of the rDNA magnification paradox in Drosophila melanogaster.

rDNA magnification in Drosophila melanogaster is defined experimentally as the ability of bb/Ybb- males to produce exceptional progeny that are wild type with respect to rDNA associated phenotypes. Here, we show that some of these bobbed-plus progeny result not from genetic reversion at the bb locus but rather from variants at two or more autosomal loci that ameliorate the bobbed phenotype of rDNA deficient males in Drosophila. In doing so we resolve several aspects of a long-standing paradox concerning the phenomenon of rDNA magnification. This problem arose from the use of two genetic assays, which were presumed to be identical, but paradoxically, produced conflicting data on both the kinetics of reversion and the stability of magnified bb+ chromosomes. We resolve this problem by demonstrating that in one assay bobbed-plus progeny arise primarily by genetic reversion at the bobbed locus, whereas in the other assay bobbed-plus progeny arise both by reversion and by an epistatic effect of autosomal modifiers on the bobbed phenotype. We further show that such modifiers can facilitate the appearance of phenotypically bobbed-plus progeny even under conditions where genetic reversion is blocked by magnification defective mutants. Finally, we present a speculative model relating the action of these modifiers to the large increases in rDNA content observed in males undergoing magnification.

Animals↗
PubMed1985

RNA directed synthesis of catalytically active Drosophila sn-glycerol-3-phosphate dehydrogenase in Xenopus oocytes.

Xenopus laevis oocytes injected with poly(A)+ RNA isolated from Drosophila melanogaster direct the synthesis of catalytically active glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8). The de novo synthesized enzyme reflects the electrophoretic properties appropriate to the stock of flies from which the injected RNA was isolated and is electrophoretically distinct from endogenous Xenopus activity. Immunoprecipitation of 35S-labeled translation products has demonstrated two immunologically related proteins with molecular masses of 32- and 34kDa which are encoded by two separate mRNA molecules. The 32-kDa protein is identical in size and charge properties to the protein purified from the fly and possesses the catalytic activity observed in the Xenopus translational assay. Poly(A)+ RNA isolated from a strain of flies bearing a CRM- null mutation at the GPDH locus does not contain translatable RNA for the 32-kDa protein. These results suggest that the two immunologically related proteins are the translational products of two separate transcripts derived from either two related loci or from differential transcription and/or processing of the same genetic locus.

Animals↗
PubMed1985

Partial purification and characterization of mitotic factors from HeLa cells.

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Alkaline Phosphatase↗
PubMed1984

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells.

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.

Amino Acid Sequence↗
PubMed1984

Thermal tolerance by embryos and larvae of the surf clam Spisula solidissima.

Thermal tolerance was tested in three developmental stages of the surf clam Spisula solidissima: cleavage stages, trochophore , and straight hinge larvae. Experiments were designed to simulate larval entrainment in the cooling system of a power plant. Eleven different temperatures were investigated from 20.2 to 43 degrees C. Exposure times varied from 1 min to 6 hr and following each of the (88) different exposures, larvae were returned to the incubation temperature for a further period of development which facilitated differentiation of dead and live animals. Pooled mortality data from triplicate experiments (for each larval state) were subjected to a stepwise regression analysis (percentage mortality on temperature and time). Equations were derived from the regression program comprising first-, second-, and third-order terms for temperature and time, and these were used to generate response surfaces relating mortality to temperature-time exposure. As with earlier work on Mulinia , the term (temperature) provides the best single predictor of larval mortality. Spisula is the only fully marine bivalve species so far subjected to this type of thermal tolerance test and is clearly the most temperature sensitive of all species investigated to date.

Animals↗
PubMed1984

Translation in Xenopus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins.

Translation of messenger RNAs (mRNAs) for human epidermal growth factor receptor (EGF-R) proteins was accomplished in a Xenopus oocyte translation system. Translation of these mRNAs in rabbit reticulocyte lysates and wheat germ extracts were unsuccessful, in accordance with the general difficulty in translating mRNAs for membrane-bound glycoproteins in conventional heterologous cell-free systems. Total poly(A)+RNA from a human epidermoid carcinoma cell line A431 was injected into oocytes from Xenopus laevis. After incubation in the presence of [35S]methionine, the oocytes were homogenized and the supernatant incubated with several different polyclonal antibodies (IgGs) against the human EGF-R. Immunocomplexes were bound to protein A-Sepharose, washed extensively, and electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. A protein of Mr = 160,000, corresponding to the EGF-R glycoprotein, was specifically precipitated from RNA-injected oocytes by each of the anti-EGF-R IgGs. This protein was not detectable in the immunoprecipitates from uninjected oocytes nor was it precipitated from RNA-injected oocytes by IgG from normal rabbit serum or IgG to an unrelated protein. A second protein of Mr = 100,000 was also immunoprecipitated from the homogenates of RNA-injected oocytes. This product corresponds to the EGF-R-related protein, present in A431 cells, which is structurally similar to the EGF-R (Weber et al., 1984). Thus, frog oocytes provide an efficient and generally applicable heterologous system for translation of mRNAs for large glycoproteins such as growth factor receptors. In addition, successful immunoprecipitation of translated products in this system provides a method for assay of mRNAs where biological activity of the proteins cannot be monitored.

Animals↗
PubMed1984

Inactivation of mitotic factors by ultraviolet irradiation of HeLa cells in mitosis.

Extracts from mitotic HeLa cells, when injected into fully grown Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) indicated by germinal vesicle breakdown (GVBD) and chromosome condensation. Recently, we observed that the MPA of mitotic cell extracts is neutralized by the inhibitors of mitotic factors (IMF) in HeLa cells, which are activated at telophase and remain active throughout the G1 period. The activity of the IMF coincides with the process of chromosome decondensation, which begins at telophase and continues until the beginning of S phase, when chromatin reaches its most decondensed state. The objective of the present study was to investigate whether these two phenomena - chromosome decondensation and the activation of IMF - were related. The activity of IMF was measured in N2O-blocked mitotic HeLa cells, in which chromosome decondensation was induced by exposure to ultraviolet light, and subsequent incubation in medium containing inhibitors of DNA synthesis, hydroxyurea and arabinosylcytosine (araC). u.v. irradiation activated IMF was seen even at very high doses of X-irradiation. The IMF seemed to inactivate the mitotic factors directly by forming a complex that precipitated on heating at 60 degrees C for 15 min. Mg2+ or polyamines (i.e. spermine, spermidine, and putrescine), agents known to promote chromatin condensation partially restored the MPA of the u.v.-irradiated mitotic cell extracts. These results tend to support the conclusion that the IMF play a role in the decondensation of chromosomes.

Cell Fusion↗
PubMed1983

Amplification and molecular cloning of murine adenosine deaminase gene sequences.

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.

Adenosine Deaminase↗
PubMed1983

Quantitation of apolipoprotein E in rabbit sera with a competitive enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay was developed to quantitate apoE levels in normal and cholesterolemic rabbit serum. The assay can detect 1 ng of apoE and has an interassay coefficient of variation of 5.1%. The assay's antigen specificity was established by the generation of a competitive displacement curve with rabbit apoE, but not with rabbit albumin nor with rabbit apoC. Nonimmune serum was not able to produce a detectable response in the assay. Lipid-protein interactions did not interfere with the assay and color development was linear throughout the incubation time. Rabbits fed a normal diet had 5.3 +/- 0.4 mg of apoE/dl serum. Rabbits fed a diet containing 1% (w/w) cholesterol for 14 days had 86 +/- 11 mg of apoE/dl serum.

Animals↗
PubMed1983

Polyspermy inhibition in the oyster, Crassostrea virginica.

Inhibition of polyspermy is a critical response during fertilization which ensures that only one sperm nucleus will fuse with the female pronucleus to restore the diploid state. Oyster (Crassostrea virginica) eggs prevent polyspermy by a process occurring at the cell surface. However, 5 min after fertilization, there are still functional sperm receptor sites available for penetration by supernumerary sperm. Reinsemination experiments indicate that there is no decrease in the number of penetration sites during this interval. Yet, the number of sperm entering eggs is restricted to one per fertilized egg at a sperm:egg ratio of 1000:1. At a sperm:egg ratio of 10(5):1, an average of only six sperm were able to penetrate each egg. Gamete binding experiments indicate that there is a gradual decrease in the number of sperm bound to eggs starting at approximately 75 sec and continuing until all sperm are detached. Since eggs are fertilized within seconds of insemination and this process takes at least 12 min, it is considered an unlikely mechanism for the polyspermy block. There are no ultrastructural correlates to the polyspermy block nor to the unbinding of sperm, such as secretion of cortical granules or fertilization envelope formation. Based on reinsemination experiments, kinetic data, and ultrastructural observations, we suggest a physiological block to polyspermy which prevents fusion of gamete plasma membranes, and is mediated by an inhibitory effect directly upon the sperm.

Animals↗
PubMed1983

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells.

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.

Animals↗