Efficacy of meloxicam in a pain model in sheep.

OBJECTIVE: To evaluate the efficacy of the non-steroidal anti-inflammatory drug, meloxicam, in alleviating pain and inflammation and on production-related variables in a model of sterile acute inflammation in sheep. METHODS: Groups of 12 mature Merino ewes received 0, 0.5, 1.0 or 1.5 mg/kg meloxicam subcutaneously 90 min before injection of 0.1 mL turpentine subcutaneously on the anterior aspect of the proximal phalanx of a forelimb. Pain- and inflammation-related variables were assessed at -18, 3, 6, 9, 12, 24, 48 and 72 h relative to meloxicam administration. Daily feed intake and body weight change 7 days later were also assessed. Pain-related variables measured were weight borne on each forelimb, lameness score, time each forelimb was raised in a 20-s interval and tolerance to a noxious mechanical stimulus. Inflammation-related variables measured were skin temperature, limb circumference, body temperature, plasma haptoglobin concentration and peripheral blood leucocyte parameters. RESULTS: Meloxicam was effective in improving all pain-related variables. A dose-dependent response was seen between 0 and 1.0 mg/kg, with no additional benefit provided by 1.5 mg/kg. At a dose rate of 1.0 mg/kg, meloxicam improved weight borne on the turpentine-treated limb by 14%, reduced the time the treated limb was held in a non-weight-bearing posture by 46%, reduced the lameness score by 58% and improved tolerance to pressure by 52%. No significant effects of meloxicam on inflammatory variables or appetite were observed. CONCLUSIONS: Using a validated pain model, the data suggested that 1.0 mg/kg meloxicam provided significant analgesic benefits to sheep.

Multisite randomised controlled trial to evaluate polypropylene clips applied to the breech of lambs as an alternative to mulesing. I: effects on body weight, breech bare area measurements and scores, wrinkle scores and faecal and urine staining.

OBJECTIVE: To assess the effects of application of occlusive polypropylene clips to lambs on body weight, breech bare area measurements and scores, wrinkle scores, and faecal and urine stain scores. PROCEDURES: A randomised controlled trial using 32,028 lambs was conducted on 208 properties across Australia. Polypropylene clips were applied at lamb marking. At each site, 160 lambs were weighed, measured for breech bare area and scored for bare area, wrinkle, dag and urine staining, and skin type and thickness. Lambs were allocated to a control (no clips) or treatment (breech and tail clips) group. Lambs were assessed on days 14 (range 10-19) and 55 (range 34-129) after clip application for body weight, breech bare area measurements and scores. On day 55 the operators also scored wrinkling and urine staining. RESULTS: At an average of 55 days after treatment, treated ewe and wether lambs had 16% and 21% greater horizontal bare area measurements, and 31.7% and 32.7% higher bare area scores than control lambs, respectively. The ewes and wether lambs also had lower wrinkle (6.8% and 5.8%, respectively) and dag scores (12% and 12.3%, respectively) than controls. Treated ewes had lower urine stain scores (18.8%) than controls. However, body weight was slightly lower in clip-treated lambs compared with controls by 0.320 kg (1.2%) and 0.430 kg (1.6%) for ewes and wethers, respectively. CONCLUSION: Polypropylene clips applied to the breech and tail of lambs increased breech bare area and reduced dag, urine and wrinkle scores. Improvements in these measures of factors that predispose to blowfly strike suggest that the application of clips may reduce the risk of breech flystrike.

Effect on the breech and tail characteristics of Merino lambs of varying the duration of application of occlusive plastic clips as an alternative to mulesing.

OBJECTIVE: The objective of the study was to determine the minimum duration of clip application required to increase the size of the perineal and tail bare areas and reduce breech wrinkle, dag or urine stain scores of Merino lambs. METHOD: The study was conducted on a single farm in western New South Wales using 491 unmulesed lambs of a plain body type (9-14 weeks of age; mean (±SD) bodyweight 26.9 ± 5.0 kg; 82% wrinkle score 1 or 2) randomly allocated into 10 groups. Groups 1-9 included 39-46 lambs treated with the clips, which were removed at 1-7, 10 or 14 days, respectively, after application. Group 10 comprised 53 lambs that were unclipped, unmulesed controls. Visual scores, bare area measurements and bodyweights were assessed before and at 28 and 60 days following clip application. RESULTS: Clips increased the size of the perineal and tail bare areas if left on the lambs for a minimum of 4-6 days. The increases in the size of the perineal and tail bare areas were similar to the results of previous studies. This indicates that the clips performed as expected, despite the current study being conducted on lambs with a plain body type. CONCLUSION: When used as an alternative to mulesing, clips should remain on lambs for a minimum of 4-6 days to effectively increase the size of the perineal and tail bare areas. Further research is required to determine the degree of protection against flystrike provided by the clip method.

Effectiveness of a non-surgical alternative to the Mules operation in sheep.

OBJECTIVE: To measure changes to the perineal bare area, local tissue reaction and healing responses of young sheep, following intradermal administration of cetrimide and polyvinylpyrrolidone (PVP), with and without ethanol, to the breech and tail. METHOD: A needle-less injector was used to deposit formulations containing 40 g/L cetrimide and 30 g/L PVP (group 2) or 20 g/L cetrimide, 30 g/L PVP and 15 g/L ethanol (group 3), within the dermis of the tail and the region surrounding the perineal bare breech area of groups (N = 8) of Merino weaner sheep. The dimensions of the perineal bare area (length, width and diagonal distances left and right) and tail width were recorded before and at intervals after treatment for 60 days. Observations of swelling and bruising and scab formation at the treatment sites were recorded for up to 35 days after treatment. Rectal temperatures were monitored for up to 35 days after treatment and bodyweight for up to 60 days after treatment. An untreated control group (group 1) was included. RESULTS: Comparison of day -3 and day 35 measurement data showed that both treated groups had significantly (P < 0.05) wider breech bare areas compared to the untreated controls and that group 2 sheep had significantly (P < 0.05) longer breech bare areas compared to group 3 sheep or to the untreated controls, which were not significantly different. At this time scabs were still firmly in place on many treated sheep. At day 35 there was no increase in tail bare area caused by either treatment. By day 60 there was no significant difference between the treated and control groups in either the breech or tail regions indicating that the changes present at day 35, were not permanent. Mean weight gain in the groups throughout the 60-day interval was unaffected by treatment. Intradermal treatment was associated with a significant elevation in body temperature. This effect lasted for 3 days and was associated with signs of discomfort and depressed appearance in at least some of the treated sheep. Bruising was mild to severe in all treated sheep within two days of treatment but was not evident in any sheep by day 21. Mild to moderate swelling was also associated with treatment but was not uniform across sheep in the groups. The tail of one sheep was severely swollen for several days. Swelling remained obvious in most treated sheep until day 14 but was not present at day 21. CONCLUSION: Under the conditions of this study intradermal injection of cetrimide had no permanent effect on bare area measurements on the breech or the amount of wool-bearing skin on the tail. It also caused signs of discomfort and pain that raise welfare concerns.

Assessment of welfare of suckling lambs following intradermal injection of cetrimide as a non-surgical alternative to conventional mulesing.

OBJECTIVE: To assess in suckling lambs the impact of intradermal injection of cetrimide, a quaternary ammonium compound formulated to induce non-surgical mulesing, on some physiological and behavioural indicators of welfare. PROCEDURES: We allocated 32 suckling lambs (9-11 weeks old) to three groups: (1) control (n = 10), (2) conventional surgical mules (n = 11) and (3) non-surgical mules (n = 11). Non-surgical mulesing was induced by intradermal injection of 4% (w/w) cetrimide + 3% (w/w) polyvinylpyrrolidone in water. Lambs were run in pens of four together with their dams. Haematology, cortisol, beta-endorphin and haptoglobin levels, and rectal temperature were monitored at least daily for the first 7 days after treatment, then weekly until day 28. Body weight was measured weekly and behaviour was measured every 15 min for 12 h on the day of treatment, then on days 1, 2, 4, 6, 12, 21 and 28 following treatment. RESULTS: The intradermal treatment induced local tissue swelling, systemic signs of severe inflammation, including high fever (> 41.0 degrees C) and elevated blood cortisol levels, by 12 h. Rectal temperatures were significantly elevated until 6 days after treatment, cortisol levels were elevated until 4 days after treatment, haptoglobin levels for at least 7 days after treatment and the neutrophil to lymphocyte ratio until 5 days after treatment. Peak cortisol values were comparable in mulesed lambs and lambs receiving the intradermal treatment, whereas the areas under the curves for cortisol and temperature were greater in lambs receiving the intradermal treatment than in mulesed lambs. Beta-endorphin levels were significantly elevated in mulesed sheep at 12 h. There was no effect of intradermal treatment on average daily gain, fibre diameter or beta-endorphin concentration. Mulesed lambs spent 44% of the time in abnormal behaviours (hunched standing, stiff walking, pawing, lateral lying and lying intention) on the day of treatment. On the day after treatment, lambs receiving the intradermal treatment spent 11% of the time (comparable to mulesed lambs) in abnormal behaviours. In comparison, control lambs spent 0.4% of their time in abnormal behaviours on the same day. CONCLUSIONS: The welfare of suckling lambs that were non-surgically mulesed by intradermal injection of cetrimide was measurably poorer than control lambs.

Effect of the non-steroidal anti-inflammatory drug, carprofen, on weaned sheep following non-surgical mulesing by intradermal injection of cetrimide.

OBJECTIVE: To assess in weaned lambs the palliative effects of the non-steroidal anti-inflammatory drug, carprofen, following intradermal injection of cetrimide to induce non-surgical mulesing. PROCEDURES: We allocated 40 weaned lambs (20-22 weeks old) to four groups of 10 animals: (1) control, 2) conventional surgical mules, (3) intradermal treatment and (4) intradermal treatment + carprofen. Non-surgical mulesing was induced by intradermal injection of 4% (w/w) cetrimide + 3% (w/w) polyvinylpyrrolidone in water. In group 4, carprofen (4 mg/kg, SC) was administered 1 h before intradermal treatment. Five weaners, including an animal from each treatment, were run in each pen. Neutrophil to lymphocyte ratio, cortisol, beta-endorphin and haptoglobin levels and rectal temperature were monitored at least daily for the first 7 days after treatment, then weekly until day 28. Body weight was measured weekly and behaviour was measured every 15 min for 12 h on the day of treatment, then on days 1, 2, 4, 6, 12, 21 and 28 following treatment. RESULTS: The intradermal treatment resulted in high fever and elevated blood cortisol by 12 h. Rectal temperatures were significantly elevated until 5 days after treatment, cortisol was elevated until 3 days after treatment, haptoglobin for at least 7 days after treatment and the neutrophil to lymphocyte ratio until 4 days after treatment. Average daily gain was depressed in the week following treatment. Abnormal behaviours (hunched standing, stiff walking, pawing, lateral lying and lying intention) were increased on the day of treatment and for 6 days post treatment. Carprofen reduced the time spent in abnormal behaviours by approximately two-thirds but did not ameliorate the physiological responses to the intradermal treatment. CONCLUSIONS: In weaner sheep, carprofen ameliorated the behavioural responses, but was unable to provide relief from the intense and sustained physiological responses to non-surgical mulesing by intradermal injection of cetrimide. Systemic side-effects may be unavoidable with formulations based on quaternary ammonium compounds that are designed to reduce the risk of fly strike in sheep by remodelling breech tissue through induction of tissue necrosis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.