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Biomedical subjects

J B Lloyd

Publications and source records attributed to J B Lloyd.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1985

pH-profile of cystine and glutamate transport in normal and cystinotic human fibroblasts.

In the human recessive condition cystinosis, cystine transport has been reported to be normal in the plasma membrane but defective in the lysosome membrane. A possible explanation is that the transport systems at the two cellular sites are identical and that the defect in cystinosis affects the porter's ability to operate at the low pH of the lysosome. To test this hypothesis the uptake of 3H-labelled cystine and glutamate by normal and cystinotic human skin fibroblasts has been measured in vitro at pH 5.8, 6.5, 7.0, 7.4 and 8.0. Uptake of glutamate was more rapid than that of cystine. Uptake of cystine increased with increasing pH, but uptake of glutamate showed no marked pH-dependence. Transport in cystinotic cells was similar to that in normal cells, and similarly affected by pH. This finding is incompatible with the hypothesis proposed above. It is concluded that the cystine porters of the plasma membrane and the lysosome membrane are probably genetically distinct.

Biological Transport, Active↗
PubMed1985

The role of pinocytosis in the cellular uptake of an amino acid.

Uptake of L- and D-alanine by the rat visceral yolk sac has been studied in vitro in incubations of short duration. It is concluded that much of the uptake of D-alanine is due to fluid-phase pinocytosis and that the plasma membrane L-alanine porter is more stereospecific than has hitherto been supposed.

Alanine↗
PubMed1985

A study of the low beta-galactosidase activity in cystinotic fibroblasts: effects of cysteamine.

beta-Galactosidase activity but not beta-glucuronidase, N-acetyl-beta-D-galactosaminidase or arylsulphatase A activity, is known to be significantly lower in cultured human skin fibroblasts from patients with cystinosis than in cells from control subjects. Incubation of cell homogenates with disulphide or thiol compounds did not affect beta-galactosidase activity, suggesting that decreased beta-galactosidase activity in affected cells was not caused by the presence of inhibiting substances or absence of activating substances. Incubating cells with 0.5 or 1.0 mmol/l cysteamine, a substance used in the clinical treatment of cystinosis because it depletes cells of excess cystine, greatly decreased beta-galactosidase activity in both cystinotic and normal cells. This effect is shown to result from enzyme instability in lysosomes with raised pH and increased thiol concentration. Thus, cysteamine, although effective in depleting cystinotic cells of excess cystine, may have the undesired side-effect of severely decreasing lysosomal beta-galactosidase.

Cells, Cultured↗
PubMed1985

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity.

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.

2,4-Dinitrophenol↗
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↗
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

Phagocytic uptake of latex beads by rat peritoneal macrophages: comparison of morphological and radiochemical assay methods.

Contrary to previous reports, commercially available 1000-nm latex beads were found to be labelable with 125I, yielding a product that retained its radiolabel on storage at 4 degrees C and when incubated in tissue-culture media. This finding permitted a radiochemical method to measure phagocytic uptake of latex particles by rat peritoneal macrophages cultured in vitro, and a direct comparison with the established method of particle counting by light microscopy. The two methods yielded closely similar data, demonstrating that the (much more convenient) radiochemical method for quantitating phagocytic uptake is both feasible and reliable. The kinetics of phagocytic uptake of the latex particles and the effect of low temperature and metabolic inhibitors (sodium fluoride and 2,4-dinitrophenol) are described. Ongoing phagocytosis did not alter the rate of fluid-phase pinocytosis by macrophages.

Animals↗
PubMed1983

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates.

The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.

Animals↗
PubMed1983

Targeting of N-(2-hydroxypropyl)methacrylamide copolymers to liver by incorporation of galactose residues.

Soluble synthetic polymers have potential as targetable carriers of pharmacological agents. Here we report that incorporation into poly[N-(2-hydroxypropyl)methacrylamide)] of an oligopeptide side-chain terminating in galactose enhanced the polymer's pinocytic uptake from the rat bloodstream by the liver. Within the liver lysosomes enzymic digestion led to the intracellular release of a drug analogue also bound to oligopeptide side-chains of the polymer.

Acrylamides↗
PubMed1983

The role of microtubules in pinocytosis. Inhibition of fluid-phase pinocytosis in the rat visceral yolk sac by mitoclasic and related agents.

Colchicine, demecolcine and vinblastine all effectively inhibited the pinocytic capture of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs cultured in vitro. Complete inhibition did not occur until 2-3h after addition of mitoclasic agent and this delay appeared to be related to the mechanism of drug-microtubule interaction rather than any difficulty of drug permeation into cells. Inhibition of pinocytosis by demecolcine was reversible, whereas inhibition by colchicine or vinblastine was not. In terms of dose-response demecolcine was slightly more effective than colchicine, but both were considerably more active than trimethylcolchicinic acid. Of the nine benzimidazoles tested for ability to inhibit pinocytosis, four severely retarded uptake and three were partially inhibitory.

Animals↗