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D A Wright

Publications and source records attributed to D A Wright.

At least 55 records · Page 3Linked to original sources
PubMed1992

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.

Antibodies, Monoclonal↗
PubMed1991

Multiple forms of maturation-promoting factor in unfertilized Xenopus eggs.

Maturation-promoting factor (MPF), which is functionally defined by its ability to induce frog oocyte maturation independent of protein synthesis, is hypothesized to be the mitotic inducer in eukaryotic cells. Previous studies have demonstrated that the cdc2 protein kinase complex (p34cdc2-cyclin) meets the criteria for MPF. In the present study, we show that MPF activity in extracts of unfertilized Xenopus eggs can be resolved into three fractions by Q-Sepharose chromatography. Of the total MPF activity recovered, approximately 20% was in the flow-through fraction that was accounted for by the cdc2 kinase complex, approximately 40% was in the 0.2 M NaCl eluate, and the remaining approximately 40% was in the 0.5 M NaCl eluate. Neither eluate contained cdc2 kinase, but each could activate cdc2 kinase upon microinjection into Xenopus oocytes. The MPF activity in the two eluates, but not in the flow-through fraction, could be depleted by the mitosis-specific monoclonal antibody MPM-2. This antibody has been shown to inhibit Xenopus oocyte maturation and deplete MPF activity from mature oocyte extract but does not recognize the cdc2 kinase complex. The three MPFs differed in apparent molecular size, H1 kinase activity, and stability at 4 degrees C. We propose that MPF activity in unfertilized Xenopus eggs resides in at least three different molecular species, the combined activities of which may be required for autoamplification of MPF.

Amino Acid Sequence↗
PubMed1991

Effect of mercury on unidirectional sodium and calcium influx in Asellus aquaticus.

The effect of mercuric chloride (HgCl2) and monomethyl mercury chloride (CH3HgCl) on unidirectional 22Na and 45Ca influx were measured in the freshwater isopod Asellus aquaticus. Flux measurements involved short-term (20 min) exposure to 22Na or 45Ca following 2 h pre-exposure to Hg. Experiments were conducted at two different Na and Ca concentrations, 0.025 mmol L-1 and 0.25 mmol L-1. HgCl2 and CH3HgCl inhibited Na influx at both Na concentrations although Na influx at 0.25 mmol L-1 was always higher. This reflected the uptake kinetics of the Na pump which was determined to be saturable with a kmax values of 32 mumol Na g-1 h-1 and a Km value of 0.8 mmol L-1. CH3HgCl generally resulted in relatively greater inhibition of Na influx. At 0.025 mmol Na L-1 all CH3HgCl concentrations tested were inhibitory to Na influx, i.e., the lowest observed effect concentration (LOEC) was less than 0.04 mumol L-1. Ca influx was inhibited by HgCl2 at all concentrations tested (LOEC less than 0.04). The degree of inhibition was unaffected by Ca concentration, which was seen as evidence for non-competitive inhibition. However, CH3HgCl, which inhibited Ca influx from 0.025 mmol Ca L-1 by greater than 68% at molar Hg concentrations greater than or equal to 1 mumol L-1 showed no significant inhibitory affect at 0.25 mmol Ca L-1. Elimination of CH3HgCl inhibition at the higher Ca concentration suggests competition in this case, possibly at the level of access to the Ca pump.

Animals↗
PubMed1991

A novel M phase-specific H1 kinase recognized by the mitosis-specific monoclonal antibody MPM-2.

At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca2(+)- and cyclic nucleotide-independent histone H1 kinase activity, referred to as growth-associated or M phase-specific H1 kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the p34cdc2 protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific H1 kinase activity. When mature Xenopus oocyte extract prepared with ATP gamma S and NaF was fractionated by gel filtration, two prominent peaks of H1 kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the p34cdc2 protein and was immobilized by the suc 1 gene product p13 and anti-cyclin B2, which are specific for the cdc2 kinase complex. However, the 600-kDa kinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific H1 kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway.

Animals↗
PubMed1991

Carbohydrate feedings 1 h before exercise improves cycling performance.

The effects of consuming two different amounts of liquid carbohydrate 1 h before exercise on the metabolic responses during exercise and on exercise performance were determined. Subjects consumed either 1.1 g (LC) or 2.2 g (HC) carbohydrate/kg body mass or a placebo (P). Subjects cycled at 70% of maximal oxygen consumption (VO2max) for 90 min and then underwent a performance trial. Blood glucose and insulin responses during exercise were different among the three trials. Total carbohydrate oxidation was greater for the carbohydrate trials compared with P. Time-trial performance was significantly improved by LC and HC. Despite elevated insulin concentrations at the start of and during exercise, and despite an initial drop in blood glucose, consumption of between 1.1 and 2.2 g liquid carbohydrate/kg body mass 60 min before moderately intense prolonged exercise can improve performance, presumably via enhanced carbohydrate oxidation.

Adult↗
PubMed1991

Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene.

We have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression (Z. Chen, M. L. Harless, D. A. Wright, and R. E. Kellems, Mol. Cell. Biol. 10:4555-4564, 1990). Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase III promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. We identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, we have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of our findings, we hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

Adenosine Deaminase↗
PubMed1991

Carbohydrate feedings before, during, or in combination improve cycling endurance performance.

This study examined the effects of no carbohydrate (PP), preexercise carbohydrate feeding (CP), carbohydrate feedings during exercise (PC), and the combination of carbohydrate feedings before and during exercise (CC) on the metabolic responses during exercise and on exercise performance. Nine well-trained cyclists exercised at 70% of maximal O2 uptake until exhaustion. Blood glucose peaked 30 min after the preexercise carbohydrate feeding and at the start of exercise was 25% below the prefeeding concentration (4.76 mM). At exhaustion, glucose had declined to 3.8 (PP), 4.0 (CP), 4.6 (PC), and 5.0 mM (CC). Insulin was 300% above basal (7 microU/ml) at the start of exercise for CC and CP and returned to baseline by 120 min of exercise. When carbohydrates were consumed, the rate of carbohydrate oxidation was significantly higher throughout exercise than during PP. Total work produced during exercise was 19-46% (P less than 0.05) higher when carbohydrates were consumed. Time to exhaustion was 44% (CC), 32% (PC), and 18% (CP) greater than PP (201 min; P less than 0.05). Performance was improved by ingestion of carbohydrates before and/or during exercise; performance was further improved by their combination. This is probably the result of enhanced carbohydrate oxidation, especially during the later stages of exercise.

Adult↗
PubMed1990

Selective activation of the N-glycosylation apparatus in uteri by estrogen.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional level.

Animals↗
PubMed1990

Removal of 2',3'-dideoxynucleotide residues from injected DNA in Xenopus laevis oocytes.

Dideoxynucleotides have proved to be potent differential inhibitors of DNA polymerases in vitro and in vivo. Used extensively in studies of DNA repair and replication, they have more recently been used as antiviral agents particularly in treating patients for acquired immunodeficiency syndrome (AIDS). Once incorporated, these sugar-modified analogues prevent the further extension of the polynucleotide chain because of the absence of a 3'-hydroxyl group. We demonstrated that, upon injection into Xenopus laevis oocytes, 2',3'-dideoxynucleotides are efficiently removed from plasmid DNA preterminated with these analogues allowing subsequent closure by ligation. The removal process is not sensitive to aphidicolin but is quantitatively inhibited by novobiocin.

Animals↗
PubMed1990

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.

Animals↗
PubMed1990

Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene.

We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

Adenosine Deaminase↗
PubMed1990

Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene.

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

Adenosine Deaminase↗
PubMed1990

Safety of 4-T MR imaging: study of effects on developing frog embryos.

Fertilized eggs of Rana pipiens (leopard frogs) were exposed to 0.15- or 4.5-T magnetic resonance (MR) imaging and were compared with unexposed fertilized eggs with respect to the percentage of embryos cleaving and percentage of tail buds forming normally. Similarly exposed or unexposed sperm were used to fertilize unexposed eggs, and the same parameters were observed. There was no evidence that MR imaging exposure at 0.15 or 4.5 T had any effect on cleaving embryos or tail bud formation. Since embryogenesis in amphibians is very sensitive to foreign insult, the authors conclude that exposure to a magnetic field of up to 4.5 T has no adverse effect on early development.

Animals↗
PubMed1989

Effects of tributyltin compounds on ionic regulation and gill ATPase activity in estuarine fish.

1. Striped bass (Morone saxatilis) were exposed to 0, 0.10, 0.34, or 1.09 micrograms/l tributyltin (TBT) for 14 days. Gill Mg2+ ATPase and serum Na+, K+, Ca2+ and Mg2+ were not altered. Na+K+ATPase was significantly (P less than 0.05) increased (+48%) at 0.10 microgram/l. 2. In striped bass gill homogenates exposed to TBT in vitro, there was significant inhibition of Na+K+ATPase at 106.0 micrograms/l (17.2%) and Mg2+ATPase at 53.0 and 106.0 micrograms/l (16.0% and 24.3%, respectively). 3. In mummichog (Fundulus heteroclitus) gill homogenates, there was significant inhibition of Na+K+ATPase at 25.3 and 50.5 micrograms/l (10.9% and 16.1%) and Mg2+ATPase at 5.1, 25.3, and 50.5 micrograms/l (26.7%, 32.2%, and 36.2%).

Animals↗
PubMed1989

Mitosis-specific monoclonal antibody MPM-2 inhibits Xenopus oocyte maturation and depletes maturation-promoting activity.

MPM-2, a monoclonal antibody specific for cells in mitosis, recognizes a family of proteins that share a common phosphorylated epitope. In this study we have shown that during the maturation of Xenopus laevis oocytes induced by progesterone, phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity. When MPM-2 (0.7-1.4 micrograms per oocyte) was injected into oocytes prior to progesterone stimulation, MPF activity failed to appear and induction of maturation was inhibited as judged by both germinal-vesicle breakdown and white-spot formation. Further, MPM-2 was able to neutralize as well as immunodeplete MPF activity from mitotic HeLa cell and mature oocyte extracts. These results suggest that MPM-2 recognizes either MPF itself or a protein(s) that regulates MPF activity and that the kinase that phosphorylates MPM-2 antigens may be a key component in the regulation of M-phase induction.

Animals↗
PubMed1989

Posttranscriptional regulation of a class of human cytomegalovirus phosphoproteins encoded by an early transcription unit.

We have further characterized and determined the origins of a family of nuclear phosphoproteins of 84, 50, 43, and 34 kilodaltons (kDa) encoded by a class of early transcripts arising from the adjacent EcoRI fragments R and d (map units 0.682 to 0.713) of the strain AD169 human cytomegalovirus genome. These RNAs have a complex spliced structure with common 5' and internal exons and alternative 3' exons with coterminal 3' ends. At early times, two fully processed species of 2.1 and 2.2 kilobases (kb) predominated. As the infection progressed to late times, there was a decrease in splicing of the RNA, generating larger transcripts 2.5 to 2.65 kb in size, which corresponded to the species which had spliced out only the first intron, as well as the completely unspliced transcript. We previously reported that the 34-kDa protein could be derived from a transcript which had failed to splice out the first intron (D. A. Wright, S. I. Staprans, and D. H. Spector, J. Virol. 62:331-340, 1988), but the origin of the other proteins was unclear. cDNA cloning has shown that the 2.1-, 2.2-, and 2.5-kb RNAs encode the 50-, 43-, and 84-kDa proteins, respectively. The shift in the splicing pattern of these RNAs with time revealed a posttranscriptional control mechanism which results in the differential accumulation of individual proteins within this family of nuclear phosphoproteins. Expression of the 84-, 43-, and 34-kDa proteins correlated well with the steady-state concentrations of their respective mRNAs. The 50-kDa protein, however, was not expressed in abundance until late times, despite the presence of the 2.1-kb mRNA in the cytoplasm at early times, suggesting a secondary level of posttranscriptional regulation for this protein. Full expression of the RNAs and proteins was dependent on continuing viral DNA synthesis. Accumulation of the 50-kDa protein was found to be particularly sensitive to the state of viral DNA replication and could not be detected after inhibition of replication. Further analysis of these proteins revealed that each one had a unique pattern of serine phosphorylation. Although there was one common site of phosphorylation, most likely located within the amino-terminal shared region, even this site showed quantitative differences in the level of phosphorylation for each of the proteins. Analysis of the Towne strain and two recent independent clinical isolates of human cytomegalovirus has shown that this family of proteins is highly conserved among human cytomegaloviruses.

Biological Evolution↗
PubMed1989

Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos.

By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.

Animals↗