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Biomedical subjects

J B Lloyd

Publications and source records attributed to J B Lloyd.

At least 91 records · Page 5Linked to original sources
PubMed1983

Inhibition of proteolysis in rat yolk sac as a cause of teratogenesis. Effects of leupeptin in vitro and in vivo.

Conceptuses from 9.5-day pregnant rats were cultured for 48 h in heat-inactivated homologous serum to which leupeptin, a specific inhibitor of the lysosomal cysteine-proteinases, was added for the final or the penultimate 6 h. The presence of leupeptin (25 micrograms/ml or above) increased the protein content of yolk sacs at harvesting to approximately twice the control value. The protein content of the embryo at harvesting was lower than that of controls. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. The presence of leupeptin did not affect the rate of uptake of the radiolabelled macromolecule by the yolk sac, nor facilitate its entry into the embryo. When formaldehyde-denatured 125I-labelled bovine serum albumin was added to the culture medium for the final 6 h of culture, little radioactivity was found in the yolk sac at harvesting, and barely any was found in the embryo. Trichloroacetic acid-soluble radioactivity was found in the culture serum. The presence of leupeptin sharply increased the levels of radioactivity in the yolk sac (but not the embryo) and sharply decreased the acid-soluble radioactivity of the culture medium. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in both yolk sac and embryo at harvesting. The presence of leupeptin increased the amount found in the yolk sac and decreased that found in the embryo. These results are interpreted as follows. Leupeptin enters the lysosomes of the yolk sac, inhibiting their cysteine proteinases. The digestion of proteins pinocytosed by the yolk sac is consequently inhibited, resulting in the accumulation of protein by the yolk sac and a decreased flow of amino acids to the embryo. Leupeptin (50 mg/kg), injected into pregnant rats at either 8.5 days or 9.5 days of gestation, induced congenital malformation in the offspring. It is proposed that leupeptin exerts its teratogenic action by inhibiting proteolysis in the lysosomes of the yolk sac, and so depriving the developing embryo of its supply of amino acids at a critical stage of development.

Abnormalities, Drug-Induced↗
PubMed1983

Evidence that protein ingested by the rat visceral yolk sac yields amino acids for synthesis of embryonic protein.

[3H]Leucine-labelled haemoglobin was prepared from rat reticulocytes incubated in the presence of [3H]leucine. Conceptuses from 9-5-day pregnant rats were incubated in vitro tox 48 h, with [3H]leucine-labelled haemoglobin present for the final 12, 8, 4, 2 or 0.5 hours. Radioactivity accumulated in visceral yolk sac and in embryonic tissue. When exposure to labelled haemoglobin was for only a short period before harvesting, all the radioactivity found in the embryo and most of that found in the visceral yolk sac trichloroacetic acid-soluble (i.e. associated with free amino acid rather than with protein). After longer exposures the proportion of radioactivity that was acid-soluble decreased to minimum values of about 20%. SDS-polyacrylamide gel electrophoresis of the protein-associated radioactivity in visceral yolk sac and embryo was performed. After exposure to labelled haemoglobin for 1 h only prior to harvesting, the yolk sac contained a single peak of radioactivity coincident in mobility with haemoglobin. The embryo contained no protein-associated radioactivity. After exposure to labelled haemoglobin for 12h, many protein bands in both yolk sac and embryo were radiolabelled. Thus a single radiolabelled protein pinocytically captured by the visceral yolk sac can give rise to the presence of many labelled proteins in embryo and visceral yolk sac. These results indicate that the source protein underwent proteolytic digestion and that the amino acids generated were re-utilized for protein synthesis in both embryonic and visceral yolk-sac cells.

Amino Acids↗
PubMed1982

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage.

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and 125I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[125I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of 125I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of Mr 120 000 was internalized by macrophages somewhat more rapidly than copolymer of Mr 46 000, but was excluded from the yolk sac.

2,4-Dinitrophenol↗
PubMed1982

Pinocytosis of poly (alpha, beta-(N-2-hydroxyethyl))-DL-aspartamide and a tyramine derivative by rat visceral yolk sacs cultured in vitro. Ability of phenolic residues to enhance the rate of pinocytic capture of a macromolecule.

Incorporation of 20% tyramine residues into its structure greatly increased the rate of pinocytosis of poly(alpha, beta-(N-2-hydroxyethyl))-DL-aspartamide (PHEA) by rat visceral yolk sacs cultured in vitro. Both the parent macromolecule and the tyramine derivative (PHEA-tyramine) were captured by adsorptive pinocytosis, the higher affinity of the derivative for the yolk sac plasma membrane being responsible for its greater rate of capture. Using 125I-labelled PHEA-tyramine, the relationship between substrate concentration and rate of capture was determined, it was also shown that following internalization, the PHEA-tyramine linkage is resistant to intracellular hydrolysis. Fluorescence micrographs were consistent with capture of both substrates being by pinocytosis and illustrated the highly efficient concentration of the tyramine derivative by yolk sac endodermal cells.

Animals↗
PubMed1982

Role of thiols in degradation of proteins by cathepsins.

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.

Acetylglucosaminidase↗
PubMed1982

Evidence for intracellular digestion of an exogenous protein by rat peritoneal macrophages.

The differential effects of lower incubation temperature on the digestion of formaldehyde-denatured 125I-labelled bovine serum albumin by intact rat peritoneal macrophages and by macrophage homogenates were studied. Digestion by intact macrophages was abolished at 20 degrees C and below, but a near-linear relationship between digestion rate and temperature between 37 degrees C and 5 degrees C was found with homogenates. Several known inhibitors of pinocytosis were found to inhibit the generation of digestion products from 125I-labelled albumin by macrophages. It is concluded that pinocytosis is an obligatory step in the catabolism of exogenous proteins by macrophages.

Animals↗
PubMed1982

Degradation of side-chains of N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal thiol-proteinases.

N-(2-Hydroxypropyl)methacrylamide copolymers bearing oligopeptide side-chains terminating in p-nitroaniline (NAp) were incubated with rat liver lysosomal enzymes in the presence of the thiol glutathione, and the rate of p-nitroaniline release was measured. Twelve of the 16 side-chains investigated were hydrolysed to release p-nitroaniline and in all but one case degradation was partially or totally inhibited by leupeptin. The effect of substrate concentration on the degradation of the most readily cleaved side-chain, -Ala-Gly-Val-Phe-NAp, was measured.

Acrylamides↗
PubMed1982

A spectrometric study of the fluorescence detection of fecal urobilinoids.

Fluorescence spectra of extracts of fecal material, in the presence of zinc ion, display the well-known green fluorescence used in the Schlesinger test for the urobilinoids, together with several other fluorescences. All of these can be demonstrated in a single spectrum by the synchronous fluorescence technique, which, with new extraction conditions, enables the urobilinoid fluorescence to be detected in quantities of human fecal material down to about 50 ng in mass. This represents a reduction on the order of a thousandfold in the detection limit of the original visual technique; selectivity too is greatly increased. The effects caused by a variety of reagents on the fluorescences have been examined. It is shown that in Edelman's version of the test the presence of mercuric ion largely quenches the fluorescence. The synchronous spectra vary considerably between different samples of human feces and between different mammals according to their diet. The specificity of the recommended technique is discussed with reference to the spectra given by other body materials.

Acetates↗
PubMed1981

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers. A potential drug delivery system.

Synthetic 125I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [125I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.

2,4-Dinitrophenol↗
PubMed1981

Effect of molecular size of 125I-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages.

Rates of pinocytosis of different molecular-weight distributions of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs and rat peritoneal macrophages were measured in vitro. Four preparations of mean molecular weights 50 000, 84 000, 700 000 and 7 000 000, were used. Macrophages captured the highest-molecular-weight preparation more rapidly than the other preparations. In contrast, rate of capture by the yolk sac decreased with increasing molecular weight. Incubations with a very-high-molecular-weight fraction derived from the 7 000 000-average-mol. wt. preparation clearly demonstrated that very large polymer molecules are not accumulated by the yolk sac, but are preferentially captured by macrophages. Analysis of the 125I-labelled poly(vinylpyrrolidone) internalized by the two cell types confirmed that low-molecular-weight material is preferred by the yolk sac, whereas the macrophage is less discriminating.

Animals↗
PubMed1981

Serum-dependence of fluid-phase pinocytosis and specificity in adsorptive pinocytosis of simple proteins in rat peritoneal macrophages.

An increase in the concentration of serum resulted in a very marked increase in the rate of fluid-phase pinocytosis in rat peritoneal macrophages, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. In contrast, the rate of uptake of colloidal [198Au]gold, an adsorptive substrate, decreased when the serum concentration was increased. This lessened uptake of colloidal gold must be due to competition by serum components. Studies on the specificity of pinocytosis of simple proteins, using 125I-labelled H4 and M4 forms of porcine lactate dehydrogenase, and formaldehyde-denatured and untreated bovine serum albumin as substrates, suggest that positive charge and hydrophobicity determine adsorptive pinocytosis of simple proteins in peritoneal macrophages. The rate of fluid-phase pinocytosis and the specificity of adsorptive pinocytosis in peritoneal macrophages are very similar to those reported for Kupffer cells.

Adsorption↗
PubMed1981

Uptake by rat peritoneal macrophages of 125I-labelled polyvinylpyrrolidone entrapped within liposomes.

Rat peritoneal macrophages in vitro capture 125I-labelled polyvinylpyrrolidone entrapped within either negatively or positively charged liposomes more rapidly than they do the free macromolecule. The uptake of negatively charged liposomes was linear with time over 10 h, whilst the uptake of positively charged ones, although more rapid, was more transient. Neither type of liposome was taken up in the presence of 2,4-dinitrophenol (100 microgram/ml), and 5 mM calcium chloride increased the uptake of negatively charged liposomes. The enhanced uptake of 125I-labelled polyvinylpyrrolidone when presented in liposomes must have been a consequence of entrapment rather than of a simple interaction between lipid and polyvinylpyrrolidone since the presence of the lipids employed or of empty liposomes had no effect on the uptake of unentrapped 125I-labelled polyvinylpyrrolidone.

Animals↗
PubMed1981

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis.

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of 125I-labeled PVP and colloidal 198Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [14C]DIVEMA and 125I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.

Animals↗