Search PubMed⌕ Search

Biomedical subjects

D J Armstrong

Publications and source records attributed to D J Armstrong.

At least 37 records · Page 2Linked to original sources
PubMed1996

Poly-(D,L-lactic acid) microspheres incorporating histological dyes for intra-pulmonary histopathological investigations.

Polylactic acid (PLA) microspheres incorporating fluorescein as a histological marker have been prepared and used as a model for the testing of inhaled PLA microspheres (2-5 microns) in the lung. PLA microspheres (20 mg) were delivered to rabbits in the form of a saline nebulization. The distribution pattern within the pulmonary system showed that the fluorescein-labelled microspheres were distributed about the four lobes in discrete groups. The comparative numbers of these groups showed a trend towards a reduced concentration in the lower lobes. Subsequent histological examination revealed that the microsphere-treated lungs had been significantly damaged after 24 h. Histological damage was assessed in terms of pulmonary haemorrhage, eosinophilia and neutrophil infiltration.

Administration, Inhalation↗
PubMed1995

Human papillomavirus type 6 E7 protein is a substrate in vitro of protein kinase C.

The E7 proteins of 'low-risk' and 'high-risk' human papillomaviruses (HPV) are phosphorylated by casein kinase II. In this study, we report that the 'low-risk' HPV 6 E7 protein, but not the 'high-risk' HPV 16 E7 protein, can be phosphorylated in vitro on threonine at amino acid position 7 by protein kinase C. This is the first example of a qualitative biochemical difference between the HPV 6 E7 and HPV 16 E7 proteins.

Amino Acid Sequence↗
PubMed1993

The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues.

In the presence of sodium dodecyl sulfate and 2-mercaptoethanol, the human papillomavirus 16 E7 protein migrates as a 17 kD protein during polyacrylamide gel electrophoresis. However, the theoretical molecular mass of this protein is approximately 11 kD. Substitution of 2 basic amino acids for 2 acidic residues in the amino terminus of the protein restored normal electrophoretic mobility. Furthermore, neutralization of negative charge through chemical modification of the wild type protein normalized migration. These results indicate that the substantial net negative charge of the wild type E7 protein is responsible for its anomalous electrophoretic behavior.

Amino Acid Sequence↗
PubMed1992

Mutagenesis of human papillomavirus types 6 and 16 E7 open reading frames alters the electrophoretic mobility of the expressed proteins.

The E7 open reading frames of human papillomavirus type 6 (HPV-6) and HPV-16 encode proteins consisting of 98 amino acids that are quite similar in sequence yet different in electrophoretic mobility. Moreover, these proteins vary strikingly in oncogenicity. To investigate the molecular basis of the differences in structure and function, site-directed mutagenesis was used to exchange non-conserved amino acid residues between the two proteins. The mutated coding regions were expressed as fusion proteins in Escherichia coli and identified by Western blotting. Comparative analysis of the affinity-purified mutated E7 fusion proteins in polyacrylamide slab mini-gels in the presence of SDS and 2-mercaptoethanol revealed altered electrophoretic mobilities. This analysis suggests that the aspartic acid at residue 4 (Asp 4) contributes to the characteristic aberrant migration of the HPV-16 E7 protein in SDS-polyacrylamide gels.

Amino Acid Sequence↗
PubMed1991

Identification of minor C18 triene and conjugated diene isomers in hydrogenated soybean oil and margarine by GC-MI-FT-IR spectroscopy.

Vegetable oils are partially hydrogenated in order to produce palatable products of suitable plasticity. The constituents of these new dietary products are complex mixtures of fatty acid isomers with different nutritional properties. A rapid method is described for separating and identifying fatty acid methyl ester (FAME) isomers of linolenic (octadecatrienoic, 18:3) acid and of conjugated octadecadienoic (18:2) acid, minor species found in hydrogenated soybean oil and margarine, by capillary gas chromatography-matrix isolation-Fourier transform-infrared (GC-MI-FT-IR) spectroscopy. FAMEs of 18:3 acid isomers in margarine, soybean oil hydrogenated in our laboratory, and isomerized linolenic acid were identified by this method, and MI-FT-IR spectra of FAME geometric isomers of octadecatrienoic and conjugated octadecadienoic acids are reported for the first time. Five major C18 triene GC peaks are found in chromatograms of isomerized methyl linolenate, representing species with tri-cis and tri-trans configurations and three species with cis-trans mixed configurations. FAME isomers with these configurations are also found for a hydrogenated soybean oil having an iodine value of 111. Methyl linolenate (tri-cis) is no longer found when soybean oil is further hydrogenated to an iodine value of 96. IR spectra characteristic of a tri-trans isomer are obtained for two test samples with iodine values of 111 and 96. Besides methyl linolenate, only isomers with a mono-trans di-cis configuration are found for the margarine analyzed. Conjugated cis-trans and trans-trans 18:2 FAME isomers are also found in all the hydrogenated soybean oil and margarine analyzed.

Chromatography, Gas↗
PubMed1991

MDL 72222 (a selective 5-HT3 receptor antagonist) prevents stimulation of intrapulmonary C fibres by pulmonary embolization in anaesthetized rabbits.

We have investigated the effects of the intravenous administration of 5-hydroxytryptamine upon the discharge frequency of pulmonary stretch receptors, irritant receptors and C fibres. Only C fibres were stimulated by this autacoid. MDL 72222 a selective 5-HT3 receptor antagonist blocks the C fibre stimulation by 5-hydroxytryptamine. It also blocks the C fibre response to miliary pulmonary embolism. These data confirm the hypothesis that 5-hydroxytryptamine is the humoral link between pulmonary embolism and tachypnoea.

Animals↗
PubMed1990

Genotypic variation in cytokinin oxidase from phaseolus callus cultures.

Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues.

Journal Article↗
PubMed1990

Phenylbiguanide not phenyldiguanide is used to evoke the pulmonary chemoreflex in anaesthetized rabbits.

The pulmonary chemoreflexes evoked by phenylbiguanide (PBG), phenyldiguanide (PDG), phenylguanidine (PG) and 5-hydroxytryptamine (5-HT) have been investigated in the pentobarbitone-anaesthetized rabbit. The rank order of potency was 5-HT greater than PBG greater than PG greater than PDG. The responses evoked by all agonists were antagonized by pre-treatment with the selective 5-HT3 receptor antagonist MDL 72222, suggesting that their reflex effects are mediated by 5-HT3 receptors located on pulmonary vagal afferents. Furthermore, considering the low potency and long injection response time of PDG compared to PBG, we conclude that previous workers have used PBG and not PDG to identify non-myelinated pulmonary vagal afferents and to evoke the pulmonary chemoreflex.

Animals↗
PubMed1990

5-Hydroxytryptamine mediates the post-embolic increase in respiratory rate in anaesthetized rabbits.

The effect of the neuronal 5-HT3 receptor antagonist MDL 72222 has been investigated on the reflex tachypnoeic response to pulmonary embolism in pentobarbitone-anaesthetized rabbits. Pre-treatment with MDL 72222 (640 micrograms kg-1, 5 min prior to embolization) significantly attenuated the reflex tachypnoeic response associated with the injection of emboli. MDL 72222 had no effect on the bradycardia associated with embolization; however, it significantly reduced the decrease in arterial blood pressure and converted the decrease in tidal volume seen in pulmonary embolism to an increase. Furthermore, MDL 72222 had no effect on the decrease in circulating platelet count associated with embolization. The data suggest that 5-hydroxytryptamine is of primary importance in mediating the post-embolic increase in respiratory rate seen in this model of pulmonary embolism.

Analysis of Variance↗
PubMed1990

The role of platelets in the reflex tachypnoeic response to miliary pulmonary embolism in anaesthetized rabbits.

Experiments were performed upon twenty-eight male Californian rabbits anaesthetized with sodium pentobarbitone and breathing room air. Successive injections of glass bead emboli (diameter 125 microns) caused an increase in respiratory rate, and decreases in tidal volume (tachypnoeic response) and platelet count. The tachypnoeic response was not observed in rabbits that had been rendered thrombocytopaenic by platelet antiserum. Induction of thrombocytopaenia by antiserum was accompanied by tachypnoea which was not significantly different (P greater than 0.05) from the response to emboli. The tachypnoeic response to antiserum, like that to emboli, was prevented by bilateral cervical vagotomy. Pre-treatment with aspirin (5 mg/kg), which inhibits the platelet-release reaction, also prevented the tachypnoeic response to both emboli and antiserum, although it did not prevent the fall in platelet count which following injection of antiserum. These data support the hypothesis that the post-embolic reflex tachypnoea is platelet dependent and may be secondary to the platelet-release reaction.

Animals↗
PubMed1989

Glycogen synthase turnover and phosphorylation in rat H4IIE hepatoma cells.

Glycogen synthase was isolated from rat H4IIE hepatoma cells by the use of specific antibodies. Immunoprecipitates from cells grown in the presence of [35S]methionine contained two 35S-labeled polypeptides, designated GS1 and GS2, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of both species was half-maximal after 3 h and remained constant up to 48 h. When cells were incubated with [32P]-phosphate, 32P was incorporated into both species with similar kinetics, half-maximal labeling occurring after 2-3 h. The steady-state ratio 32P/35S was significantly higher for the lower mobility GS2 polypeptide. Pulse-chase experiments showed that the two subunits followed similar kinetics with respect to 35S-labeling. However, the turnover of 32P on the GS2 subunit was significantly faster (t1/2 approximately 30 min) than that on the GS1 subunit (t1/2 approximately 2 h). We suggest that the two polypeptides represent different phosphorylation states of the glycogen synthase subunit and are rapidly interconverted.

Animals↗
PubMed1989

Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum.

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.

Adenine↗
PubMed1988

Cytokinin Oxidase from Phaseolus vulgaris Callus Cultures : Affinity for Concanavalin.

Cytokinin oxidase activity from Phaseolus vulgaris cv Great Northern callus cultures exhibited affinity for the lectin concanavalin A. Over 80% of the activity extracted from the callus tissue bound to a concanavalin A-Sepharose 4B column. The bound activity was eluted from the column by the addition of methylmannose to the eluting buffer. On the basis of this result, it appears that most of the cyokinin oxidase activity present in Great Northern callus cultures exists in the form of a glycoprotein. The apparent pI of this enzyme, as estimated by chromatofocusing, is approximately 5.0.

Journal Article↗
PubMed1987

Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues : Enhanced in Vitro Activity of the Enzyme in the Presence of Copper-Imidazole Complexes.

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N(6)-(Delta(2)-isopentenyl)-adenine-2,8-(3)H (i(6) Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N(6)-side chain of i(6)Ade.

Journal Article↗
PubMed1986

The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione.

Unstable metabolites may arise during the metabolism of xenobiotic compounds with enzyme systems other than the cytochrome P-450 system. This depends on the enzyme system involved and the structure of the xenobiotic compound being metabolized. Normally detoxifying pathways may transform selected chemicals into toxic metabolites. In our laboratory we have demonstrated that DBE is metabolized by both cytochrome P-450 and GSH S-transferases. Although the cytochrome P-450 metabolite is reactive and will covalently bind to protein and nucleic acid to some extent, and the GSH S-transferase system conjugates it and under conditions of low DBE exposure is able to detoxify it. In contrast, GSH S-transferase catalyzes the direct conjugation of GSH with DBE. This can result in formation of a reactive intermediate that preferentially binds to nucleic acids and is responsible for the DNA damage observed following DBE exposure. The selective toxicity of this xenobiotic compound may be due to the preponderance of activating GSH conjugating enzymes in the extrahepatic organs. However, this difference alone does not appear sufficient to explain the selection of extrahepatic organs as sites of DBE-induced toxicity.

Animals↗
PubMed1986

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern.

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N(6)-(Delta(2)-isopentenyl)adenine-8-(14)C (i(6) Ade-8-(14)C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i(6) Ade-8-(14)C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.

Journal Article↗