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Biomedical subjects

R Duncan

Publications and source records attributed to R Duncan.

At least 217 records · Page 12Linked to original sources
PubMed1985

Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification.

One to 2 h after transfer of HeLa cells into fresh serum-containing medium, when translation rates are maximal, the initiation factor proteins were examined on immunoblots of two-dimensional gels. Eukaryotic initiation factor (eIF)-2 alpha, eIF-2 beta, and eIF-4A each formed a single immunoreactive spot; eIF-2 gamma formed 2 spots; and eIF-4B formed a complex array of 12-20 spots. After 4 days of growth in unreplenished medium, when translation rates have dropped 4-6-fold, several alterations in the isoelectric forms were observed: eIF-2 alpha now occurred in 2 forms, eIF-2 beta was present in 3-4 forms, and the most acidic cluster of eIF-4B variants was decreased or absent while a new isoelectric variant appeared at the basic end of the array. No changes were observed for eIF-2 gamma or eIF-4A. The 35-50-kDa subunits of the multiprotein initiation factor eIF-3 also showed no changes when the aforementioned growth states were compared. Resolution of 32P-labeled lysates by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the eIF-2 alpha modification and the loss of eIF-4B variants reflected changes in phosphorylation states. Stimulation of 4-day grown cells with fresh serum-containing medium caused a reversal of the initiation factor modifications back to the forms prevailing shortly after replating. This analysis indicates that covalent modifications appear concurrently with decreasing initiation rates and suggests that they may be causative.

Blood↗
PubMed1985

Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates.

During growth in unreplenished medium, the fraction of active, polysomal ribosomes progressively decreases about 3-fold from 80-90% to only 20-40% due to a reduced rate of initiation. To assess whether the abundance of initiation factors could be involved in this repression of translational activity. HeLa cell cytoplasmic lysates were resolved by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and spots corresponding to the initiation factor proteins were quantitated. The relative abundance of most of the initiation factor proteins only decreases by 10-40% and roughly parallels that of the ribosomes. Measurement of the rates of synthesis and turnover of the initiation factor proteins establishes that during periods of active growth, synthesis and degradation occur coordinately with total cell protein. As growth rate decreases, the synthesis of some initiation factor proteins, particularly eukaryotic initiation factor (eIF)-3 subunits, becomes depressed. Serum stimulation of serum-depleted cells recruits most inactive ribosomes and mRNAs into polysomes, but most initiation factor mRNAs are not selectively recruited. The principal exceptions are eIF-3p24 which exhibits 4-5 fold enhanced synthesis and eIF-3p44 and eIF-4A whose syntheses are moderately stimulated.

Blood↗
PubMed1985

The mechanism of Ptychodiscus brevis toxin-induced rat vas deferens contraction.

Ptychodiscus brevis, which causes Florida red tide, produces Ptychodiscus brevis toxin (PBTX) known to contain neurotoxins and to induce rhinorrhea, tearing, and cough in normal humans and wheezing in asthmatic subjects. It was previously reported (J Allergy Clin Immunol 69:418, 1982; 73:824, 1984) that PBTX causes canine tracheal smooth muscle contraction via stimulation of sodium channels in the axons of parasympathetic postganglionic nerves and the release of acetylcholine from these nerve endings. This was postulated to be an asthma-triggering mechanism. In this article the toxins were evaluated to determine if they also stimulate sodium channels on adrenergic nerve endings and release norepinephrine. Rat vas deferens was selected as the experimental tissue. Both PBTX and norepinephrine contracted rat vas deferens. Prazosin 10(-6) mol/L blocked the response to PBTX (3 micrograms/ml) (88.3% to 27.3% contraction [n = 6; p less than 0.001]) and the response to norepinephrine (EC50 was shifted from 1.67 X 10(-6) mol/L to 1.25 X 10(-4) mol/L in the presence of prazosin 10(-6) mol/L [n = 6; p less than 0.001]). Phentolamine 10(-6) mol/L also blocked both PBTX and norepinephrine. Tetrodotoxin 10(-7) mol/L, a sodium channel blocker, completely blocked the response to PBTX but not to norepinephrine. The response to PBTX was significantly reduced from 1.53 gm of tension in controls to 0.29 gm of tension (n = 6; p = 0.002) in tissues obtained from rats pretreated with reserpine (2 mg/kg per day for 2 days, injected intraperitoneally). Verapamil 10(-5) mol/L blocked the PBTX response, and PBTX caused no contraction in calcium-free media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers.

Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in rho-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of rho-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of rho-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Gly-Phe-NAp where 5.1% of the bound rho-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.

Acrylamides↗
PubMed1985

Shutoff of host translation by encephalomyocarditis virus infection does not involve cleavage of the eucaryotic initiation factor 4F polypeptide that accompanies poliovirus infection.

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different.

Animals↗
PubMed1985

Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells.

Activation of the interferon-inducible, double-stranded RNA-dependent protein kinase was monitored in monolayer cultures of control and interferon-treated HeLa cells infected with encephalomyocarditis virus. The extent of phosphorylation in the intact cell of the alpha-subunit of eucaryotic protein synthesis initiation factor eIF2 by the kinase was determined for the first time in this type of system, using a two-dimensional immunoblot technique. Virus protein synthesis and the kinetics of activation of the ppp(A2'p)nA (n greater than or equal to 2) system were analyzed in parallel. Enhanced phosphorylation of eIF2-alpha was obvious at 9 h and increased by 12 h postinfection. ppp(A2'p)nA and ppp(A2'p)nA-mediated rRNA cleavage were observed from 6 h. No viral protein synthesis was detected in cells in which a general inhibition of protein synthesis developed with time. It can be concluded that both the kinase and ppp(A2'p)nA system are active in interferon-treated, encephalomyocarditis virus-infected HeLa cells.

Encephalomyocarditis virus↗
PubMed1985

Biological effects of soluble synthetic polymers as drug carriers.

Soluble synthetic polymers have already, with limited success, found some use medically as plasma expanders and as polymeric drugs. Currently, many other potential biomedical applications are under evaluation, including the use of soluble polymers as targetable drug carriers, as carriers of immunogenic compounds, and as bioadhesives. In order to exploit their undoubted clinical potential to the fullest, it is obviously essential to understand the inter-relationship between synthetic polymers and their biological environment. The biological effects of soluble synthetic polymers to be covered include their immunogenicity, the therapeutic potential of polymeric drugs, and polymer-drug conjugates and their biocompatibility. These active responses are discussed in relation to the differential fates of different soluble polymers in the body.

Animals↗
PubMed1984

Mechanism of interferon action. Increased phosphorylation of protein synthesis initiation factor eIF-2 alpha in interferon-treated, reovirus-infected mouse L929 fibroblasts in vitro and in vivo.

The effect of interferon (IFN) treatment and virus infection on the phosphorylation both in vitro and in vivo of the alpha subunit of protein synthesis initiation factor eIF-2 (eIF-2 alpha) was examined in mouse fibroblast L929 cells. The [gamma-32P]ATP-mediated in vitro phosphorylation of eIF-2 alpha catalyzed by cell-free extracts prepared from IFN-treated, uninfected cells was dependent upon exogenously added double-stranded RNA (dsRNA). However, the dsRNA requirement for eIF-2 alpha phosphorylation in vitro was eliminated by prior infection of cells with reovirus Dearing strain virions but not with defective top component particles. The enhanced phosphorylation in vitro of eIF-2 alpha and ribosome-associated protein P1 depended in a similar manner upon the multiplicity of virus infection. The extent of phosphorylation in vivo of eIF-2 alpha prepared from L929 cells was also examined by utilizing two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting techniques. About 5-10% of the eIF-2 alpha was typically phosphorylated in vivo in untreated, mock-infected cells, whereas 25-30% was phosphorylated in IFN-treated, reovirus-infected cells. An intermediate extent of eIF-2 alpha phosphorylation, routinely between 15 and 20%, was observed with either IFN treatment or reovirus infection alone. The integrity of eIF-4A and eIF-4B was also examined by two-dimensional electrophoresis and immunoblotting, and no significant alterations in molecular size or charge heterogeneity were detected when these factors were prepared from IFN-treated, reovirus-infected cells as compared to untreated, uninfected cells.

Animals↗
PubMed1984

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation.

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.

Centrifugation, Density Gradient↗
PubMed1984

Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

2,4-Dinitrophenol↗
PubMed1984

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions.

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.

Buffers↗
PubMed1984

S6 phosphorylation accompanies recruitment of ribosomes and mRNA into polysomes in response to dichlororibofuranosyl benzimidazole.

Dichlororibofuranosyl benzimidazole (DRB), a potent inhibitor of nuclear RNA synthesis and messenger RNA (mRNA) accumulation, produces a paradoxical mobilization of rRNA and mRNA from the subpolysomal pool into polysomes in HeLa cells during the first 40 min of treatment. S6 is phosphorylated concurrently with polysome accumulation, and ribosomal subunits containing phosphorylated S6 are preferentially localized in polysomes, indicating that they form initiation complexes more readily than their non-phosphorylated counterparts.

Dichlororibofuranosylbenzimidazole↗
PubMed1984

The poly(A)(+)RNA sequence complexity is also represented in poly(A)(-)RNA in sea-urchin embryos.

The extent to which the poly(A)(+)RNA sequence complexity from sea-urchin embryos is also represented in poly(A)(-)RNA was determined by cDNA cross-hybridization. Eighty percent or more of both the cytoplasmic poly(A)(+)RNA and polysomal poly(A)(+)RNA sequences appeared in a poly(A)(-) form. In both cases, the cellular concentrations of the poly(A)(-)RNA molecules that reacted with the cDNA were similar to the concentrations of the homologous poly(A)(+) sequences. Additionally, few, if any, abundant poly(A)(+)mRNA molecules were quantitatively discriminated by polyadenylation, since the abundant poly(A)(+)sequences were also abundant in poly(A)(-)RNA. Neither degradation nor inefficient binding to oligo (dT)-cellulose can account for the observed cross-reactivity. These data indicate that, in sea-urchin embryos, the poly(A) does not regulate the utilization of mRNA by demarcating an mRNA subset that is specifically and completely polyadenylated.

Animals↗
PubMed1983

Protein synthesis eukaryotic initiation factors 4A and 4B are not altered by poliovirus infection of HeLa cells.

Infection of HeLa cells by poliovirus results in the inhibition of translation of capped cellular mRNA. A plausible mechanism for this inhibition is that the structure of one or more initiation factors involved in the recognition of capped mRNA is altered. Eukaryotic initiation factor (eIF) 4A and eIF-4B are implicated in mRNA binding to 40 S ribosomal subunits and can be cross-linked to oxidized capped mRNA. We examined these factors in HeLa cell lysates by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. No alterations in the number of molecules/cell, in the molecular size, or in extents of covalent modification were detected when lysates from infected and mock-infected cells were compared. The integrity of eIF-2 and several eIF-3 polypeptides was also examined and likewise no alterations were detected. The failure of the translational machinery to recognize capped mRNA therefore is not due to a change in the structure of these initiation factors.

Cell Transformation, Viral↗