Effect of aestivation on muscle characteristics and locomotor performance in the Green-striped burrowing frog, Cyclorana alboguttata

The Green-striped burrowing frog. Cyclorana alboguttata survives extended drought periods by burrowing underground and aestivating. These frogs remain immobile within cocoons of shed skin and Mucus during aestivation and emerge from their burrows upon heavy rains to feed and reproduce. Extended periods of immobilisation in mammals typically result in muscle atrophy and a decrease in muscle performance. We examined the effect of aestivation and hence prolonged immobilisation, on skeletal Muscle mass. in vitro muscle performance, and locomotor performance in C. alboguttata. Frogs were aestivated in soil for 3 months and were compared with control animals that remained active, were fed, and had a continual supply of water. Compared to the controls, the wet mass of the gastrocnemius. sartorius, gracilus major. semimembranosus. peroneus, extensor cruris, tibialis posticus and tibialis anticus longus of aestivators remained unchanged indicating no muscle atrophy. The in-vitro performance characteristics of the gastroenemius muscle were maintained and burst swimming speed Was Unaffected, requiring no recovery from the extended period of immobilisation associated with aestivation. This preservation of muscle size, contractile condition and locomotor performance through aestivation enables C. alboguttata to compress their life history into unpredictable windows of opportunity, whenever heavy rains occur.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

Investigation of in vitro transdermal absorption of fentanyl from patches placed on skin samples obtained from various anatomic regions of dogs

Objective-To investigate in vitro transdermal absorption of fentanyl from patches through skin samples obtained from various anatomic regions of dogs. Sample Population-Skin samples from 5 Greyhounds. Procedure-Skin samples from the dogs' thoracic, neck, and groin regions were collected postmortem and frozen. After samples were thawed, circular sections were cut and placed in Franz-type diffusion cells in a water bath (32degreesC). A commercial fentanyl patch, attached to an acetate strip with a circular hole, was applied to each skin sample. Cellulose strips were used as control membranes. Samples of receptor fluid in the diffusion cells were collected at intervals for 48 hours, and fentanyl concentrations were analyzed by use of high-performance liquid chromatography. Results-Mean +/- SD release rate of fentanyl from the patch, defined by its absorption rate through the non-rate-limiting cellulose membrane, was linear during the first 8 hours (2.01 +/- 0.05 pg/cm(2) of cellulose membrane/h) and then decreased. Fentanyl passed through skin from the groin region at a faster rate and with a significantly shorter lag time, compared with findings in neck or thoracic skin samples. Conclusions and Clinical Relevance-In vitro, fentanyl from a patch was absorbed more quickly and to a greater extent through skin collected from the groin region of dogs, compared with skin samples from the thoracic and neck regions. Placement of fentanyl patches in the groin region of dogs may decrease the lag time to achieve analgesia perioperatively; however, in vivo studies are necessary to confirm these findings.

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of anin vitroculture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Analysis and application of an equilibrium model for in vitro bioassay systems with three components: Receptor, hormone and hormone-binding-protein

A simple theoretical framework is presented for bioassay studies using three component in vitro systems. An equilibrium model is used to derive equations useful for predicting changes in biological response after addition of hormone-binding-protein or as a consequence of increased hormone affinity. Sets of possible solutions for receptor occupancy and binding protein occupancy are found for typical values of receptor and binding protein affinity constants. Unique equilibrium solutions are dictated by the initial condition of total hormone concentration. According to the occupancy theory of drug action, increasing the affinity of a hormone for its receptor will result in a proportional increase in biological potency. However, the three component model predicts that the magnitude of increase in biological potency will be a small fraction of the proportional increase in affinity. With typical initial conditions a two-fold increase in hormone affinity for its receptor is predicted to result in only a 33% increase in biological response. Under the same conditions an Ii-fold increase in hormone affinity for receptor would be needed to produce a two-fold increase in biological potency. Some currently used bioassay systems may be unrecognized three component systems and gross errors in biopotency estimates will result if the effect of binding protein is not calculated. An algorithm derived from the three component model is used to predict changes in biological response after addition of binding protein to in vitro systems. The algorithm is tested by application to a published data set from an experimental study in an in vitro system (Lim et al., 1990, Endocrinology 127, 1287-1291). Predicted changes show good agreement (within 8%) with experimental observations. (C) 1998 Academic Press Limited.

A computational study of shock speeds in high-performance shock tubes

This paper describes U2DE, a finite-volume code that numerically solves the Euler equations. The code was used to perform multi-dimensional simulations of the gradual opening of a primary diaphragm in a shock tube. From the simulations, the speed of the developing shock wave was recorded and compared with other estimates. The ability of U2DE to compute shock speed was confirmed by comparing numerical results with the analytic solution for an ideal shock tube. For high initial pressure ratios across the diaphragm, previous experiments have shown that the measured shock speed can exceed the shock speed predicted by one-dimensional models. The shock speeds computed with the present multi-dimensional simulation were higher than those estimated by previous one-dimensional models and, thus, were closer to the experimental measurements. This indicates that multi-dimensional flow effects were partly responsible for the relatively high shock speeds measured in the experiments.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

In vitro human epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 from a range of solvents

Purpose. To study epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 (BP) from a range of single solvent vehicles and evaluate solvent effects on permeability parameters. Methods. The solubility of BP was measured in a number of solvents. Penetration of BP across human epidermis and high density polyethylene (HDPE) membranes was studied from 50% saturated solutions in each solvent. Results. Maximal BP fluxes from the solvents across the two membranes varied widely. Highest fluxes were observed from 90% ethanol (EtOH) for epidermis and from isopropyl myristate (IPM) and C12-15 benzoate alcohols (C12-15 BA) for HDPE membrane. Both the flux and estimated permeability coefficient and skin-vehicle partitioning of BP appeared to be related to the vehicle solubility parameter (delta(v)). The major effects of solvents on BP flux appear to be via changes in BP diffusivity through the membranes. Conclusions. Minimal penetration of sunscreens such as BP is best achieved by choosing vehicles with a delta(v) substantially different to the solubility parameter of the membrane.

Tetraphyllidean metacestodes of teleosts of the Great Barrier Reef, and the use of in vitro cultivation to identify them

The tetraphyllidean metacestode diversity of 310 teleost fishes, including 87 species from 31 families, was examined from Heron Island, The Great Barrier Reef, Australia. Eleven metacestode 'types' were identified with the use of light microscopy. Host-specificity varied greatly among metacestode types. Incorporation of in vitro cultivation allowed generic identification for some types. Types 1 and 2 belong to Uncibilocularis Southwell, 1925, and have triloculate bothridia and one pair of Forked hooks with unequal prongs; Type 3 has quadriloculate bothridia. Hook development was insufficient to determine in which genus, Acanthobothrium van Beneden, 1849 or Calliobothrium van Beneden, 1850, this type may belong. Type 4 has unilocular bothridia with simple edges and belongs to Anthobothrium van Beneden, 1850. Type 5 has multiloculated bothridia which are invaginated within pouches. This type belongs to the Rhinebothriinae although its generic identity cannot be determined. The bothridia of Type 5 everted within 24 hours of in vitro cultivation and revealed the presence of two forms, one having 48 loculi per bothridium, the other 72 per bothridium. In vitro studies provide additional support for existing theories of onchobothriid scolex development.