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Biomedical subjects

J B Lloyd

Publications and source records attributed to J B Lloyd.

At least 19 recordsLinked to original sources
PubMed2000

Lysosome membrane permeability: implications for drug delivery.

The membrane of the lysosome contains substrate-specific porters for a wide range of metabolites. Their physiological role is in promoting the efflux of the products of intralysosomal catabolism. With few exceptions, the specificity of these porters makes them unlikely candidates for the translocation of xenobiotics across the lysosome membrane. Where efflux from the lysosome is possible, it is likely to be accomplished by passive diffusion. Experimental studies on passive diffusion across the lysosome membrane have shown that its characteristics are similar to those of other biological membranes. Ease of permeation decreases with increasing hydrophilicity. Macromolecules and some highly hydrophilic molecules as small as sucrose are effectively non-permeant. The notional hydrogen-bonding capacity of molecules (an inverse correlate of oil:water partition coefficient) has been found a good predictor of permeance. Predictions of ease of permeation across lysosome membranes is of value when drug delivery strategies are contemplated that involve a drug-conjugate reaching the lysosome compartment and drug release there by the lysosomal enzymes. These strategies will be unsuccessful if the drug is unable to leave the lysosome and reach the cellular sites where its pharmacological action is required.

Animals↗
PubMed2000

Radiolabeling, stability, and body distribution in rats, of low molecular weight polylactide homopolymer and polylactide-polyethyleneglycol copolymer.

In order to study its fate in vivo, a low molecular-weight polylactide homopolymer was derivatized with a p-methoxyphenyl moiety, so as to make it susceptible to radiolabeling with 125I. A low molecular weight polylactide-polyethyleneglycol copolymer capped with ap-methoxyphenyl residue was also synthesized. The derivatized polymers were successfully [125I]iodinated in organic medium. The radiolabeled products were freed from [125I]iodide by dialysis and shown to be stable for 24 h on incubation at 37 degrees C in buffered saline or in blood. On longer incubation at 37 degrees C in buffered saline the radiolabeled polylactide released [125I]iodide and [125I]iodinated 3-(p-methoxyphenyl)propionic acid. The radiolabeled copolymer was more stable on incubation at 37 degrees C in buffered saline, but some [125I]iodide was released. The tissue distribution of radioactivity was determined 5 min, 1, 5 and 24 h after injecting male rats with 125I-labeled homopolymer or copolymer. Intravenous, intraperitoneal and subcutaneous injection routes were employed. Further rats were injected with [125I]iodide, to aid interpretation of the data. After administration of labeled homopolymer, a high concentration of radioactivity was found in the liver tissue. The levels slowly decreased over 24 h, and the polymer was successively found in the small and large intestine and the faeces. This is probably indicative of excretion via the bile. Concurrently radioactivity was excreted in the urine. After administration of labeled copolymer, a high concentration of radioactivity was found in the liver and the residual soft tissue, the latter fraction containing two-thirds of the radioactivity one hour after injection. The precise tissue location that this result indicates was not identified. After 1 h radioactivity was excreted in the faeces, again probably via the bile, and in the urine. Tissue distributions after intraperitoneal or subcutaneous injections were concordant with the above results and interpretations, with the additional factor of slow clearance from the injection site.

Animals↗
PubMed1999

Effects of methionine supplement on methionine incorporation in rat embryos cultured in vitro.

The effect of supplementary L-methionine (Met) on the incorporation of methionine was evaluated in 9.5-day rat conceptuses cultured in vitro. Parallel experiments with L-leucine (Leu) were performed for comparison. Conceptuses were cultured for 24 hr in the presence of 3H-labeled Met or Leu, and the incorporation of radiolabel into the embryo and visceral yolk sac was measured. Supplementary Met proportionately increased the incorporation of Met, but supplementary Leu did not have as great an effect on the incorporation of Leu. A hypothesis is presented to explain these findings. It is proposed that Met, but not Leu, is a rate-limiting nutrient for organogenesis-stage rat embryos cultured in rat serum. The results are also discussed with reference to the established efficacy of supplementary folic acid in decreasing the incidence of neural tube defects in human populations and to claims that Met reverses certain teratogenic phenomena, both in vitro and in vivo.

Animals↗
PubMed1999

Cellular uptake and release of two contrasting iron chelators.

Desferrioxamine and CP94 (1,2-diethyl-3-hydroxypyridin-4-one) are metal chelators used or proposed for use in the clinical treatment of iron overload. Recent data on their capacity to deplete intracellular iron led to the conjecture that the differences observed arose from the different membrane-penetration properties of the two compounds. The time-course of accumulation and subsequent release of [14C]CP94 by the rat visceral yolk sac in-vitro was compared with that of [14C]desferrioxamine and for 125I-labelled poly(vinylpyrrolidone), a marker for fluid-phase endocytosis. The results indicate that [14C]CP94 crosses the plasma and lysosome membranes rapidly whereas [14C]desferrioxamine and 125I-labelled poly(vinylpyrrolidone) are effectively incapable of crossing these membranes, entering cells only by endocytosis. It is concluded that although CP94 readily enters and leaves cells, desferrioxamine has the potential to accumulate to high concentration in the lysosomes and complex with intralysosomal iron. The results support and extend the proposed correlation between pharmacological activity and capacity for membrane penetration.

Animals↗
PubMed1998

Bisphenols that stimulate cells to release alkali metal cations: a structure-activity study.

The laxative action of phenolphthalein (5) is believed to result from induction of potassium and water efflux from the colon epithelium. In cultured cells, K+ efflux is promoted by 5 and by a contaminant (1) present in commercial phenol red. Six compounds with chemical structures related to those of 5 and 1 were tested for ability to induce the release of 86Rb from COS-7 cells preloaded with this isotope: 4,4'-(9-fluorenylidene)diphenol (2), 4, 4'-(9-fluorenylidene)dianiline, 4, 4'-(9-fluorenylidene)bisphenoxyethanol, 1,1'-bi-2-naphthol, 4, 4'-biphenol, and bis(4-hydroxyphenyl)methane. With one exception these compounds were all inactive at a concentration of 10 microM. However, 2 caused profound 86Rb efflux at concentrations as low as 100 nM. Concentrations of 5 1-2 orders of magnitude higher were needed to achieve similar levels of activity. The three compounds known to be active in this experimental system share a common feature that is absent in all the inactive compounds: a five-membered ring structure, one of whose carbon atoms is disubstituted with p-hydroxyphenyl residues. Because 2 and 5 are readily available, comparative studies on the mechanism of action of these biphenols at the cellular level can now be undertaken.

Animals↗
PubMed1998

Lysosome membrane permeability to anions.

The permeability of rat liver lysosomes to some inorganic and aliphatic organic anions was investigated, using an osmotic-protection methodology. Lysosomes were incubated at 25 degreesC in 250 mOsm solutions of potassium salts of the anions, in the presence of valinomycin, and the latency of lysosomal hexosaminidase measured at intervals. Lysosomes suspended in 250 mM sucrose at 25 degreesC were stable for up to 4 h. When suspended in 250 mOsm solutions of potassium salts of inorganic acids, latency was lost at rates indicating anion permeance decreasing in the order thiocyanate, nitrate and iodide>bromide>chloride>sulfate. This rank order does not correspond with the anion selectivity of any known anion transporter, and is closer to that of the lyotropic series. Results with the potassium salts of aliphatic organic acids indicate little correlation between permeation and hydrocarbon chain length, although formate was more rapidly permeant than acetate and its higher homologs. By contrast, oxalate was less permeable than other dicarboxylic acids. The presence of one or more hydroxy groups decreased permeance. A correlation between permeance and the acid's lowest pKa suggested that penetration was due principally to the entry of the undissociated acid, but there is evidence that the (much more abundant) singly charged anionic form is also significantly permeant.

Animals↗
PubMed1998

Effects of polyethyleneimine on endocytosis and lysosome stability.

Polyethyleneimine (PEI) is shown to destabilize isolated rat liver lysosomes, as indicated by a decrease in the latency of their acid N-acetyl-beta-glucosaminidase. PEI also inhibited the generation of radiolabeled digestion products from 125I-labeled bovine serum albumin endocytosed by rat visceral yolk sac in vitro. However, PEI did not greatly inhibit the endocytic uptake of a nondigestible fluid-phase substrate, fluorescein isothiocyanate (FITC)-dextran. It is hypothesized that PEI inhibits the adsorptive endocytosis of 125I-labeled bovine serum albumin, and thus its subsequent intralysosomal digestion, by competing with and displacing the labeled protein from its binding sites on the visceral yolk sac cell surface. This hypothesis suggests a plausible explanation for the ability of PEI to act as an efficient vector for gene and oligonucleotide transfer into mammalian cells. PEI present in the culture medium is carried into cells by adsorptive endocytosis. Concentrated thus on the endosome membrane, it permeabilizes this membrane and so affords DNA conjugated to the PEI an otherwise unavailable mode of access into the cytoplasm.

Acetylglucosaminidase↗
PubMed1998

Intestinal permeability to polyethyleneglycol and sugars: a re-evaluation.

1. Previous studies have indicated that the permeability of polyethyleneglycol across the human intestine is anomalously high in comparison with the permeability of sugars with similar molecular mass. In consequence it has been proposed that two or more distinct mechanisms must exist for the translocation of these classes of molecule or, alternatively, that the molecular parameter determining rate of penetration is each molecule's minimum molecular dimension. 2. The notional hydrogen-bonding capacity of a molecule correlates well with oil-water partition coefficient and also, in a variety of experimental systems, with rate of passive diffusion across biological membranes. A molecule's hydrogen-bonding capacity is calculated by inspecting the structural formula and summing the individual theory-derived hydrogen-bonding capacities of the molecule's functional groups. 3. A classic set of intestinal permeability data that includes several ethyleneglycol oligomers and several sugars is re-analysed. A good correlation between permeability and hydrogen-bonding capacity is demonstrated. Specifically, there is no discontinuity between the polyethyleneglycols and the sugars. The data are compatible with a simple model in which all the molecules studied cross the intestine by passive diffusion across cellular membranes.

Dietary Sucrose↗
PubMed1997

Lysosome membrane permeability to amines.

The permeability of rat liver lysosomes to xenobiotic organic compounds possessing nitrogen functions was investigated, using an osmotic-protection methodology. It was first shown that rat liver lysosomes are stable for at least one hour when incubated in 250 mM sucrose within the pH range 5 to 9. Primary and tertiary amines with pKa values within this pH range, and with differing numbers of aliphatic hydroxy or ether groups, were chosen for study and their permeability investigated at a range of pH values. The results indicate that uncharged amines can cross the lysosome membrane, and that the permeability of such molecules can be predicted from their total hydrogen-bonding capacity. The notional hydrogen-bonding capacity of an uncharged tri-substituted nitrogen with no attached hydrogen atom, as in pyridine or in a tertiary aliphatic amine, is deduced to be approximately 1, and that of an uncharged primary amine approximately 2. A hydrogen-bonding capacity of at least 11 is deduced for cationic nitrogen, implying that most if not all molecules containing a charged nitrogen atom cannot cross the lysosome membrane by passive diffusion. The implications for lysosome physiology and pharmacology are discussed.

Acetylglucosaminidase↗
PubMed1997

Uptake of microparticles by rat visceral yolk sac.

The visceral yolk sac (VYS) is responsible for a major part of the amino acid nutrition of the early post-implantation rat embryo and possibly also at the fetal stage of gestation. The mechanism involves endocytic uptake of proteins by the tissue's epithelial cells followed by intralysosomal digestion to amino acids. The amino acid so generated are used for protein synthesis in both the embryo and the VYS. Previous reports had indicated that the endocytic capacity of the VYS might be limited to exclude larger macromolecules. This study demonstrates that Percoll, which comprises 30-nm silica particles coated with polyvinylpyrrolidone (PVP), is as effectively captured by the 17.5-day rat VYS cultured in vitro as PVP itself. Uptake of 125I-labelled Percoll was progressive with time over 5 h and was inhibited by a low incubation temperature, 2,4-dinitrophenol (50 micrograms/ml), EGTA (5 mM), colchicine (10 micrograms/ml) or cytochalasin B (10 micrograms/ml). After uptake of 125I-labelled Percoll, VYSs released only 20 per cent of their radioactivity when re-incubated in fresh medium for 3 h. These data, and electron micrographs showing Percoll in intracellular vacuoles, are all consistent with uptake by endocytosis. Percoll's rate of uptake by the VYS indicates that, like 125I-labelled PVP, it enters the cell chiefly by fluid-phase pinocytosis. It is concluded that endocytosis by the VYS will efficiently capture even the largest globular proteins, and that previous indications of a relatively low size exclusion reflected the loosely coiled configuration of the synthetic polymers used in the earlier studies.

2,4-Dinitrophenol↗
PubMed1997

Uptake and processing of 59Fe-labelled and 125I-labelled rat transferrin by early organogenesis rat conceptuses in vitro.

The delivery of iron to the early organogenesis rat embryo has been studied, using 59Fe- and 125I-labelled rat transferrin. Rat conceptuses at 9.5 days postconception were cultured for 27 or 51 h in whole rat serum. Rat transferrin labelled with 59Fe was added for the final 0.1, 0.5, 6, 24 or 48 h of culture. Radioactivity accumulated progressively in both the embryo and the visceral yolk sac. Similar results were obtained when unconjugated 59Fe3+ was added to the rat serum used as culture medium. Both acid-soluble and acid-insoluble 59Fe were substantially present in the embryo and yolk sac after all exposure periods. When conceptuses were cultured in the presence of 125I-labelled rat transferrin, acid-soluble radioactivity was progressively released into the culture medium, but accumulation into the embryo and visceral yolk sac was slight and did not change with duration of exposure to the labelled protein. Similar findings were obtained using 125I-labelled bovine serum albumin. In these experiments, there was a close correspondence between the amount of iron accumulated by the embryo and visceral yolk sac in the final 24 h of a 51-h culture and the amount of transferrin converted into acid-soluble products in the same period. Visceral yolk sacs from 17.5-day pregnant rats were explanted and cultured in the presence of 59Fe-labelled rat transferrin, 125I-labelled rat transferrin or 125I-labelled bovine serum albumin, for periods up to 3 h. Again uptake of 59Fe increased with time of incubation, and the 125I-labelled proteins were digested to acid-soluble products which were released into the culture medium. The results indicate that transferrin delivers iron for incorporation into both the embryo and the visceral yolk sac, and are consistent with a mechanism involving receptor-mediated endocytosis of iron-laden transferrin by the cells of the visceral yolk sac. The transferrin itself appears to be quantitatively degraded, following delivery of iron to the yolk sac cells, a result that differs from findings in other cell types, in which the protein is not degraded but returns to the plasma membrane to participate in further cycles of iron acquisition and delivery.

Animals↗
PubMed1997

Leucine sources for the rat fetus.

Fetal and maternal plasma were assayed for the concentration of free leucine, acid-insoluble radioactivity and acid-soluble radioactivity at intervals after an intravenous bolus injection of [3H]leucine into anaesthetized pregnant rats at 17.5 days post-conception. The concentrations of total free leucine and of free [3H]leucine in maternal and fetal plasma were effectively unchanged from 5 to 180 min post-injection. Plasma free leucine concentrations in the fetus were five times those in the mother. The concentration of free [3H]leucine in fetal plasma was similar to that in maternal plasma. Thus the specific radioactivity of free leucine in fetal plasma is substantially lower than that in maternal plasma, indicating that a significant portion of the free leucine in plasma of the 17.5-day rat fetus comes from a source other than the free leucine in the maternal plasma. The data are consistent with a major contribution of amino acids coming from the degradation of extraembryonic protein in the yolk sac. Other possible sources of unlabelled leucine are discussed.

Animals↗
PubMed1997

Investigations into mechanisms of amino acid supply to the rat embryo using whole-embryo culture.

The technique pioneered by D.A.T. New for the in vitro culture of early post-implantation rat embryos has been used to study nutritional mechanisms during early organogenesis. The results indicate that the principal route for amino acid supply to the 8.5- to 11.5-day embryo involves the endocytosis of proteins into cells of the visceral yolk sac endoderm, their digestion in lysosomes, and transmission of the amino acids to the growing embryo. Free amino acids constitute a comparatively unimportant source. Inhibition of either endocytosis or intralysosomal proteolysis diminishes amino acid supply to the embryo, and this can result in embryonic death or maldevelopment during organogenesis.

Amino Acids↗
PubMed1996

Sources of amino acids for protein synthesis during early organogenesis in the rat. 3. Methionine incorporation.

Rat conceptuses at 9.5 days post-conception were cultured for 27 h in whole rat serum. [3H]Methionine, or rat serum proteins containing [3H]methionine, was introduced at 24 or 6 h before the termination of the culture. The total clearance of radioactivity into the embryo and the visceral yolk sac from the two sources was measured; also the extent to which the accumulated radioactivity was acid-insoluble. Similar experiments, but using [3H]leucine, were performed for comparison. The results indicate that free amino acid and protein can both serve as sources of amino acids for incorporation into the embryo and yolk sac, and it is estimated that in vivo over 95 per cent of the methionine (and the leucine) incorporated into these tissues arises from protein captured and digested by the yolk sac. Almost all the leucine accumulated into the conceptus is present as protein, but a larger fraction of the methionine accumulated is found in acid-soluble form. When the amino acids were delivered in the form of plasma proteins, the incorporation of methionine was two to three times more efficient than that of leucine, an observation most readily explained by leucine being provided in excess of requirements. In the light of reports that an adequate concentration of free methionine is important for the normal development of rat embryos in vitro, it is concluded that, although most of the amino acid required by the embryo is supplied as protein, the small fraction supplied as free amino acid may be critical for methionine but probably not for leucine.

Amino Acids↗
PubMed1996

Sources of amino acids for protein synthesis during early organogenesis in the rat. 4. Mechanisms before envelopment of the embryo by the yolk sac.

It was previously shown that uptake and digestion of protein by the visceral yolk sac supplies almost all of the amino acid needed by the 9.5-11.5-day rat conceptus cultured in vitro. Our aim was to test the hypothesis that protein uptake and digestion may not be as important as an amino acid source in the 8.5-9.5-day period, a stage of development before the yolk sac placenta envelops the embryo and before the vitelline circulation is established. Eight and a half-day rat conceptuses were cultured in serum supplemented with trace amounts of free [3H]leucine, [3H]leucine-containing serum proteins, free [3H]methionine or [3H]methionine-containing serum proteins. The incorporation of radiolabelled amino acid into acid-soluble and acid-insoluble fractions of the conceptus was determined. Leucine from either source was incorporated principally into proteins of the conceptus, but a greater proportion of the methionine incorporated was found in the low molecular weight fraction. It is estimated that 88 per cent of the leucine and 96 per cent of the methionine used by the conceptus was derived from protein in the culture serum; free amino acid comprised a minor supply source. We conclude that, despite the different anatomic relationships, the majority of amino acid incorporated into newly synthesized proteins of the conceptus very early in organogenesis is supplied by the digestion of protein in extraembryonic tissue, most likely the visceral yolk sac.

Amino Acids↗