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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
PubMed2001

Recovery of Mycobacterium avium subsp. paratuberculosis from nematode larvae cultured from the faeces of sheep with Johne's disease.

A study was conducted to determine whether trichostrongylid nematode larvae become contaminated with Mycobacterium avium subsp. paratuberculosis when they develop in the faeces of sheep with Johne's disease. Nematode larvae were hatched from ova in the faecal samples of affected sheep. Larval sheaths were removed and these as well as exsheathed larvae were subjected to radiometric culture for M. paratuberculosis. The organism was recovered from washing water used to prepare the larvae, third stage larvae and larval sheaths, but not from exsheathed larvae. The recovery of M. paratuberculosis from larvae was associated with the severity of the histological lesions in affected sheep and with the results of culture of the organism from intestinal tissues and faeces. Nematode parasites of sheep might be able to act as mechanical vectors for M. paratuberculosis as the organism associates with infective third stage larvae when these develop in the faeces of sheep with Johne's disease.

Animals↗
PubMed2001

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium.

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for the presence of the bacterium in modified BACTEC 12B medium, both before and after exposure to gamma irradiation. The water used to wash the larvae off the faecal cultures was also tested for the presence of the bacterium. Positive growth was confirmed as M. avium subspecies paratuberculosis by IS900 polymerase chain reaction and restriction endonuclease analysis of the product. M. avium subspecies paratuberculosis was detected in the unirradiated larval suspensions and wash waters of all three nematode species, and in the irradiated H. contortus larval suspension.

Animals↗
PubMed2000

In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep.

In vivo lymphocyte subset depletion offers a unique opportunity to study the roles of different cellular components of the immune system of sheep during infection with orf virus. Lambs were depleted of specific lymphocyte subsets by the intravenous administration of monoclonal antibodies against ovine lymphocyte surface markers and then challenged with orf virus. The skin lesions that developed were scored visually as to their severity. Blood samples were collected to monitor the lymphocyte depletions and to measure orf-virus-specific antibody levels. Skin biopsies were collected from the lesion site and studied to determine the course of the infection and the presence of various cell types and orf virus. All the sheep developed orf virus lesions after infection. All three of the CD4-depleted lambs were unable to clear virus from their skin and did not have an antibody response to the virus. Virus was also detected in the skin of one each of the three CD8-depleted, WC1-depleted and control sheep on the final day of the trial. CD8(+) lymphocytes did not appear to be essential for viral clearance later in the infection. Depletion of the majority of gammadelta(+) T-cells did not affect the outcome of orf virus infection. In sheep with high orf-virus-specific antibody titres at the time of infection, orf lesions healed faster than lesions in sheep with low antibody levels, and this occurred regardless of the lymphocyte depletion status of the animals. This study suggests that the presence of CD4(+) T-cells and orf-virus-specific antibodies are important for the control of viral replication in the skin of infected sheep.

Animals↗
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↗
PubMed2000

Evaluation of a laboratory test to detect resistance to closantel in Haemonchus contortus.

OBJECTIVE: To evaluate a laboratory test for closantel resistance in Haemonchus contortus. PROCEDURE: Field isolates of H contortus, known to be resistant to closantel, were tested in the assay. In addition, mixtures of closantel-susceptible and closantel-resistant laboratory reference strains were tested to develop a method of predicting the proportion of resistant worms in a sample from the field. RESULTS: The assay correctly identified as resistant all of the closantel-resistant field isolates of H contortus. It also identified one isolate with an in vivo efficacy of 98% as having emerging resistance. Testing of the mixtures of laboratory reference strains revealed that an isolate would be classified as resistant when it consists of about 25% or more resistant worms. Test samples that are not fully susceptible yet contain less than 25% resistant worms may be classified as emerging resistance. CONCLUSION: The in vitro migration assay is a sensitive method of detecting closantel resistance in H contortus.

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↗