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Biomedical subjects

R Duncan

Publications and source records attributed to R Duncan.

At least 199 records · Page 11Linked to original sources
PubMed1987

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF.

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.

Animals↗
PubMed1987

Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F.

Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.

Chromatography↗
PubMed1987

Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats.

A copolymer of N-(2-hydroxypropyl) methacrylamide (HPMA) and N-methacryloyltyrosinamide was prepared and fractionated using Sepharose 4B/6B (1:1) chromatography to produce eight HPMA copolymer fractions of narrow polydispersity and mean molecular weight (Mw) ranging from 12 to 778 kD. These fractions were radioiodinated and injected intravenously, subcutaneously, and intraperitoneally into rats. Their bloodstream-concentration profiles were monitored and rates of excretion assessed. Following intravenous administration the circulating blood volume available to the copolymers was not molecular-weight-dependent. A molecular-weight threshold limiting glomerular filtration was identified at approximately 45 kD, and preparations greater than this threshold were lost from the bloodstream only slowly by extravasation. Molecular weight did not influence the movement of copolymers from the peritoneal compartment to the bloodstream after intraperitoneal injection. The transfer rates observed could be accounted for by bulk phase lymphatic drainage alone, no transcapillary routes being implicated. Following subcutaneous administration the largest HPMA copolymer fraction (Mw = 778 kD, diameter approximately 30 nm) showed increased retention at the site of the injection, approximately 20% of the dose remaining there after 21 days. This could result from physical restriction of movement or from internalization into local phagocytic cells. The smaller copolymer fractions moved readily into the bloodstream whence they were either lost in the urine or they gradually penetrated into other tissues and organs. Long-term (21 days) body distribution of copolymers following both intraperitoneal and subcutaneous administration showed size-dependent accumulation in organs of the reticuloendothelial system.

Acrylamides↗
PubMed1987

The cross-cultural study of U.S. and Greek adolescents: blood pressure data.

Blood pressures (BP) were determined in 1409 boys aged 8-16 years at three one-year intervals in New York City (NYC) and in Greece, areas of high and low incidence of coronary heart disease (CHD), respectively. Non-Greek American boys had significantly lower systolic and diastolic BP than all the Greek boys from NYC and from Greece. The boys residing in Crete and in Athens had higher BP than all other cohorts. After covariance adjustment for height, ponderal index and age, significant differences among the cohorts showed the non-Greek American cohort to have the lowest and the Cretan cohorts the highest BP levels. The Cretans lead a lifestyle noted for the absence of other risk factors for CHD. Thus, while the incremental increase in blood pressure might have a corresponding increase in CHD risk among individuals, there are fewer individuals in Crete at elevated risk overall based on the CHD experience of the U.S. and Greece.

Adolescent↗
PubMed1987

Mapping of the large RNA genome segment of infectious pancreatic necrosis virus by hybrid arrested translation.

Segment A, the larger dsRNA segment of IPNV which encodes three of the four virus-coded polypeptides (preVP2, VP3, and NS) was cloned and physically mapped. The plus and minus RNA strands of the virus genome were separated and the A+ and B+ RNA strands were identified. A nested set of cDNA subclones, coterminal with the 5' end of A+ RNA, were used in hybrid arrested translation experiments. Hybrid arrest conditions which blocked the 5' two-thirds of A+ RNA allowed the in vitro synthesis of only VP3, while hybridization of the RNA to cDNA representing the 5' half of A+ RNA allowed the synthesis of both NS and VP3 but not of preVP2. In vitro translation of A+ RNA yielded all three polypeptides. It is, therefore, concluded that the order of the three polypeptides on A+ RNA is 5'-preVP2-NS-VP3-3'. These results imply that internal initiation of translation could take place on the RNA at least in vitro at sites located hundreds of nucleotides downstream from the first in-phase AUG codon near the 5' end.

Animals↗
PubMed1987

Lower airway smooth muscle contraction induced by Ptychodiscus brevis (red tide) toxin.

Ptychodiscus brevis toxin (PBTX) is produced by the organism Ptychodiscus brevis. This toxin causes a phenomenon that has come to be known as Florida red tide. It also stimulates neuronal sodium channels, resulting in activation of the cholinergic and adrenergic nerve endings of the autonomic nervous system in upper airway smooth muscle and rat vas deferens, respectively, as previously reported. It is the cholinergic stimulating action that has been implicated as a possible "triggering" event in bronchial asthma. This article concerns the investigation of whether PBTX may also affect lower airways and by what mechanism any contractile response to PBTX in lower airways may be induced. PBTX was found to elicit contractions in isolated canine lower airway smooth muscle. The threshold concentration was 0.15 microgram/ml, the peak response occurred at 6.0 micrograms/ml, and the concentration causing half-maximal response of the group was 0.57 microgram/ml. Pharmacologic analysis demonstrated that atropine (10(-6) mol/L) blocked the response to both PBTX and acetylcholine, tetrodotoxin (10(-7) mol/L) blocked the response to PBTX but not to acetylcholine, and verapamil (10(-5) mol/L) blocked the response to PBTX and reduced the response to acetylcholine. Four consecutive contractile responses to PBTX (3 micrograms/ml) produced rapid tachyphylaxis. The fourth contraction was 60% less than the initial response. A fifth response to PBTX after exposure to indomethacin (2.8 X 10(-6) mol/L) was increased and resulted in a contraction that was only 25% less than the initial response. The exogenous addition of prostaglandins (PG), PGE1 and PGE2, to indomethacin-treated lower airway strips selectively suppressed the contractile response to PBTX. Other PGs tested (PGA2, PGB2, PGD2, PGF2 alpha and PGI2) did not affect the PBTX response. These results indicate that PBTX produces spasm in lower airway smooth muscle and that it does this by stimulation of sodium channels in the cholinergic nerve fibers. The results also demonstrate a rapid reduction in the contractile response to PBTX. The results also demonstrate that the reduction is mediated by PGs of the E series.

Acetylcholine↗
PubMed1987

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro.

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

Acrylates↗
PubMed1987

Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions.

Full-length and truncated genome segment A-specific infectious pancreatic necrosis virus cDNA was subcloned into plasmid transcription vectors, and runoff transcripts were produced in vitro. These transcripts were translated in cell-free rabbit reticulocyte lysates and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-specific polypeptides were gel purified and mapped by partial proteolysis with N-chlorosuccinimide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide profiles were compared with those of the corresponding polypeptides purified from infectious pancreatic necrosis virus-infected cells or prepared by in vitro translation of denatured genomic RNA. The cDNA directed the synthesis of authentic pVP2, VP3, and NS polypeptides as well as a number of previously undescribed polypeptides. A 101,000-molecular-weight polypeptide was isolated and shown to be the unprocessed infectious pancreatic necrosis virus polyprotein. The NS polypeptide appears to be a virus-encoded protease responsible for the cleavage of pVP2 from the polyprotein. The carboxy terminus of NS was mapped to within three or four amino acids on the polyprotein. The most likely internal translation start sites responsible for NS and VP3 production in vitro were also mapped.

Amino Acid Sequence↗
PubMed1987

Guillain-Barré syndrome following acute head trauma.

A case of classical Guillain-Barré syndrome following acute head trauma is described. The association of Guillain-Barré syndrome with head injury per se is not well recognized, and a possible immunological explanation is proposed.

Craniocerebral Trauma↗
PubMed1986

Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopecek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518-521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0-11.6 mol%) were injected intravenously into rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80-90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosinamide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37 degrees C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.

Acrylates↗
PubMed1986

Pinocytic capture and exocytosis of rat immunoglobulin IgG-N-(2-hydroxypropyl)methacrylamide copolymer conjugates by rat visceral yolk sacs cultured in vitro.

Rat immunoglobulin (IgG) was covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via glycylglycyl spacer. The resultant conjugate, free IgG and HPMA copolymer (containing a low percentage of tyrosinamide to facilitate radiolabelling) were radioiodinated, and their rates of pinocytic uptake, intracellular degradation and exocytic release by rat visceral yolk sacs cultured in vitro were determined. Free IgG was pinocytosed rapidly by the yolk sac and some IgG was subject to intracellular proteolysis. In comparison the IgG-HPMA copolymer conjugate was captured more slowly, but faster than unmodified HPMA. IgG was also exocytosed rapidly by the yolk sac following pinocytic capture and similarly IgG-HPMA copolymer had a much higher rate of release than unmodified HPMA. Measurement of tissue accumulation of 125I-labelled IgG-HPMA copolymer in the presence of increasing concentrations of non-radiolabelled IgG showed competition for membrane binding sites between the free, and polymer-bound immunoglobulin. These experiments indicate that immunoglobulins can be covalently bound to a soluble polymer developed as a drug-carrier in such a way that they can still interact with specific membrane receptors and they are subsequently subjected to specific cellular transport mechanisms.

Animals↗
PubMed1986

The mechanism of ergonovine-induced airway smooth muscle contraction.

There have been three articles in the clinical literature of ergonovine maleate-induced bronchospasm. The effect of the alkaloid on isolated canine tracheal smooth muscle was analyzed to investigate the mechanism of ergonovine-induced airway smooth muscle contraction. Both ergonovine and 5-hydroxytryptamine (5HT, serotonin) contracted the smooth muscle preparations with EC50s of 1.35 X 10(-8) mol/L and 5.06 X 10(-7) mol/L, respectively. The maximal contractile response observed with ergonovine was approximately 30% less than that observed with 5HT. Methysergide competitively blocked both ergonovine and 5HT responses with similar calculated pKB values (8.33 against ergonovine and 8.46 against 5HT) and also similar pA2 values determined by Schild plots (8.50 and 8.45, respectively). The relative affinity and efficacy of ergonovine versus 5HT were determined by use of a concentration of the irreversible antagonist, phenoxybenzamine, which partially blocked receptor sites. The calculated affinity of ergonovine was about 16 times higher than that of 5HT. The relative efficacy at EC100 for ergonovine was 0.2, but at EC10 it was 41.9 (5HT efficacy = 1). Ergonovine 10(-9) or 10(-8) mol/L shifted the 5HT dose-response curve to the right without reducing the maximal response, but the shift was nonparallel. Blockade of muscarinic (atropine), alpha 1-adrenergic (prazosin), beta-adrenergic (propranolol), H1 (pyrilamine), or H2 (cimetidine) receptors did not alter ergonovine-induced contraction. These data indicate that ergonovine directly contracts canine tracheal smooth muscle as a result of its combination with 5HT receptors. This effect may result in precipitation of an asthmatic attack in susceptible individuals.

Animals↗
PubMed1985

Characterization of the adsorptive pinocytic capture of a polyaspartamide modified by the incorporation of tyramine residues.

Previously it has been shown (Duncan, R., Starling, D., Rypácek, F., Drobník, J. and Lloyd, J.B. (1982) Biochim. Biophys. Acta 717, 248-254) that incorporation of tyramine residues into poly (alpha, beta-(N-2-hydroxyethyl]-DL-aspartamide (PHEA) greatly increases its rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro. Here we describe the relationship between the tyramine content (1.2-21.9 mol%) of modified PHEA and its rate of uptake by yolk sacs. Above a level of substitution of approximately 10 mol% the rate of uptake rises rapidly, and the concentration-dependence of capture is indicative of uptake by adsorptive pinocytosis. Serum proteins were shown to compete effectively for membrane binding sites, indicating a nonspecific interaction of PHEA-derivatives with the yolk sac membrane. PHEA derivatives of the same tyramine content, but of different mean molecular weights (Mr), were captured at the same rates.

Adsorption↗