Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae.

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.

Difference spectroscopic characterisation of the cytochrome complement in Acanthocheilonema viteae.

(Dithionite-reduced) minus (ferricyanide-oxidised) difference spectra of 600 x g and 12,000 x g subcellular pellet fractions of adult male Acanthocheilonema viteae exhibited alpha-absorption maxima (296 K) attributable to Cyt c555, Cyt b562 and aa3 (600-605 nm). The gamma(Soret) maximum of both fractions was evident at 427 nm, with a shoulder at 432-434 nm. 600 x g and 12,000 x g pellet fractions of adult female and mixed-sex adult A. viteae exhibited similar absorption maxima. (Succinate-reduced)--(ferricyanide-oxidised) difference spectra of the 12,000 x g pellet fraction of mixed-sex adult A. viteae showed absorption maxima at 555 and 562 nm, 600 and 630 nm, suggesting the reduction of Cyt c555, Cyt b562, Cyt aa3 (600 nm) and an unidentified species (630 nm peak) Antimycin A (10(-6) M) induced the disappearance of the maxima at 555, 600 and 630 nm corresponding to Cyt c555, Cyt aa3 and the unidentified species; the maximum at 562 nm prevailed in the presence of antimycin A. These antimycin A induced changes can be cited as classical evidence for the functional involvement of these a, b and c type cytochromes in respiratory electron transport. (Dithionite reduced + CO)--(dithionite reduced) difference spectra suggest that adult A. viteae may have one or more CO-binding-species, one of which appears to be a low-spin-haemoprotein with a b-type or c-type haem, which has essentially an electron carrier function rather than a ligand binding function.

The occurrence of ecdysteroids in the cestode, Moniezia expansa.

The occurrence of free ecdysteroids in the sheep cestode, Moniezia expansa, was demonstrated. Significant amounts of conjugated ecdysteroids were not detected. Characterization of the free hormones by high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by gas chromatography/mass spectrometry (selected ion monitoring) indicated the presence of ecdysone, 20-hydroxyecdysone and 20,26-dihydroxyecdysone. Analysis of the ecdysteroids by radioimmunoassay in segments along part of the strobila indicated that the anterior parts contained the greatest amount of hormone. GC/MS (SIM) analysis of the hormones in a strobilar segment containing the most mature proglottids suggested the presence of several ecdysteroid metabolites.

Ecdysteroids in adults of the nematode, Dirofilaria immitis.

Adult males and females of the dog heartworm, Dirofilaria immitis, were extracted separately and, following separation of the free and conjugated ecdysteroid fractions, the conjugates were hydrolysed enzymically. Both the ecdysteroids released by hydrolysis of the conjugates and the free hormones were further purified and analysed by a combination of radioimmunoassay, thin-layer chromatography and high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). Both males and females contained free and conjugated ecdysteroids. Evidence was obtained for the presence of ecdysone, 20-hydroxyecdysone, 20,26-dihydroxyecdysone and possibly ponasterone A. The possible parallel between ecdysteroid endocrinology in nematodes and insects is discussed.

Effect of nedocromil sodium on neutrophil and eosinophil-induced epithelial cell desquamation in a human in vitro epithelial model.

A human amniotic epithelial membrane preparation was used as a model to study epithelial responses to neutrophils and eosinophils, obtained from normal subjects using Percoll density gradient methods, and activated by phorbyl myristate acetate (PMA). Incubation of activated neutrophils with the epithelial membrane resulted in epithelial cell desquamation, probably due to release of proteases. Activated eosinophils resulted in epithelial cell damage but no desquamation, an action that appeared to be mimicked by major basic protein (MBP). The addition of nedocromil sodium did not significantly inhibit neutrophil-induced epithelial cell desquamation. In one preliminary experiment, nedocromil sodium (10(-4) to 10(-5) mol/L) inhibited epithelial cell desquamation induced by a mixed population of eosinophils and neutrophils.

Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.

Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.

Strongyloides ratti: mitochondrial enzyme activities of the classical electron transport pathway in the infective (L3) larvae.

Submitochondrial particles prepared from S. ratti L3 larvae exhibited NADH-oxidase (NOX), NADH-ferricyanide reductase (NFR), NADH-cytochrome-c-reductase (NCR), succinate-cytochrome-c-reductase (SCR), and cytochrome-aa3-oxidase activities of 2.1 +/- 0.3, 8.9 +/- 1.3, 0.6 +/- 0.1., 1.0 +/- 0.2 and 1.2 +/- 0.3 nm min-1 mg protein-1 respectively, at 37 degrees C. The NCR and NOX activities were 39.3% and 23.5% of the NFR activity, suggesting the occurrence of a rate-limiting step or bifurcation of the respiratory electron transport (RET) pathway on the oxygen-side of RET-Complex I. The NCR activity was 50% that of cytochrome-aa3-oxidase activity which suggests partitioning of electron flow at the level of RET-Complex III and/or the quinone-function. Antimycin A and rotenone but not 2-thenoyl trifluoroacetone (TTFA) inhibited NCR activity, the EC50 values were 3.6 x 10(-6) M, 3.7 x 10(-7) M, respectively. SCR activity was inhibited by antimycin A (EC50 = 3.8 x 10(-6) M) and TTFA (EC50 = 2.8 x 10(-5) M) but not by rotenone. The results suggest that presence of classical and alternate RET-pathways in S. ratti L3 larvae.

The effect of electron transport (ET) inhibitors and thiabendazole on the fumarate reductase (FR) and succinate dehydrogenase (SDH) of Strongyloides ratti infective (L3) larvae.

The fumarate reductase (FR) and succinate dehydrogenase (SDH) activities of isolated submitochondrial particles (SMPs) prepared from axenised L3 larvae of S. ratti were characterised with respect to their response to a selected range of inhibitors. Rotenone (a specific inhibitor of electron transport Complex I) inhibited the S. ratti FR (EC50 = 3.0 x 10(-7) M) but not SDH. This strongly suggests that the S. ratti FR is functionally linked with the S. ratti ET-Complex I. 2-Thenoyltrifluoroacetone (TTFA, an inhibitor of ET-Complex II) inhibited FR (EC50 = 2.6 x 10(-5) M) and SDH (EC50 = 2.8 x 10(-5) M) with similar effectiveness. Sodium malonate (substrate analogue of succinate) had a greater affinity for SDH (EC50 = 6.8 x 10(-4) M), than FR (EC50 = 1.9 x 10(-2) M). Sodium fumarate was ca. 8-fold more effective in inhibiting the S. ratti FR (EC50 = 6.0 x 10(-4) M) than SDH (EC50 = 4.8 x 10(-3) M). The S. ratti FR was more sensitive to inhibition by thiabendazole (TBZ; EC50 = 4.6 x 10(-4) M) than SDH (EC50 > 1.0 x 10(-3) M), suggesting that one of the sites-of-action of TBZ to be the FR of S. ratti mitochondria. More potent inhibitors of S. ratti FR, if developed, may prove to be effective chemotherapeutic agents in the management of human strongloidiasis.

Respiration in the filarial nematode Brugia pahangi.

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.

Strongyloides ratti: fumarate reductase and succinate dehydrogenase activities of infective larvae.

Submitochondrial particles prepared from axenised infective (L3) larvae of S. ratti (homogonic-strain) were assayed spectrophotometrically for fumarate reductase (FR) and succinate dehydrogenase (SDH) and their kinetic properties characterised. The S. ratti FR (pH 8.2; 37 degrees C) exhibited a maximum specific activity of 3.45 nmol (min)-1 (mg protein)-1 at a sodium fumarate concentration of 0.3 mM. Interestingly, the FR activity declined at fumarate concentrations greater than 0.3 mM. The mechanism of this unusual inhibitory effect requires further study. The S. ratti SDH (pH 8.2; 37 degrees C) showed a Vmax of 17.4 nmol (min)-1 (mg protein)-1; the Kmsucc was 0.5 mM. Although the SDH:FR ratio cannot predicate vectorial electron flow as would occur in vivo, an in vitro ratio of 5.04:1 was observed for SMPs derived from S. ratti L3 larvae.

An alternative protocol for the serial passage and maintenance of a homogonic strain of Strongyloides ratti in female Sprague-Dawley rats.

A method which does not involve the tedious use of watch glass coprocultures for obtaining filariform infective (L3) larvae of Strongyloides ratti from faecal pellets of infected Sprague-Dawley rats is described. The alternative method utilises Baermannization (18 h) of faecal pellets to yield rhabditiform (L1) larvae of S. ratti and their subsequent culture for 72 h at 19 degrees C in tissue-culture-flasks containing only dechlorinated tap water to yield infective filariform (L3) larvae. The yields and infectivity of the L3 larvae obtained from the two methods were essentially similar.

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport.

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO2) of 181.8 +/- 12.4 ng atoms min-1 mg dry weight-1 at 35 degrees C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 microM) produced 33 +/- 6.5% inhibition of the E-QO2. Unusually the rotenone-induced inhibition was not relieved by 5 mM-succinate. The E-QO2 of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 microM; 4 microM-antimycin inhibited less than or equal to 10% of the E-QO2. The electron donor couple ascorbate/TMPD augmented the E-QO2 in the presence of rotenone (2 microM) and antimycin A (4 microM) by 110%. Azide (1 mM) stimulated the antimycin A refractory QO2 by 36.6 +/- 7.2% which was only partially inhibited by 1.0 mM-KCN (IC50 = 0.8 mM). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.

Murine strongyloidiasis: the effects of cyclosporin A and thiabendazole administered singly and in combination.

A single Cyclosporin A (CsA) dose of 30 mg kg-1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 +/- 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 +/- 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg-1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 +/- 4.1% and 69.0 +/- 9.6%, respectively. Orally administered CsA was less effective than 5 mg TBZ kg-1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg-1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated.

The efficacy of two electron transport inhibitors (720C80 and 993C76) on murine strongyloidiasis: a comparison with albendazole.

The clinical efficacy of albendazole (ABZ) in the treatment of chronic uncomplicated strongyloidiasis has been reported to be highly variable. In our murine model of strongyloidiasis a single oral dose of 5 and 10 mg kg-1 ABZ reduced (at day 4 post infection) the faecal larval count (FLC) by 54.2 +/- 12.5% and 81.5 +/- 10.2%, respectively. 100 mg kg-1 ABZ reduced the FLC by 100%. Two inhibitors of protozoan and filarial electron transport (720C80 and 993C76) inhibited the endogenous O2 consumption of intact infective (L3) larvae of S. ratti by > 50% at 2 x 10(-5) M in vitro, and reduced the FLC by 72 +/- 9.3% and 62.0 +/- 10.3% respectively in vivo, at a dose of 70 mg kg-1. These results suggest that compounds designed as selective inhibitors of protozoan electron transport have significant efficacy against murine strongyloidiasis and may prove useful in the management of human strongyloidiasis.

Typing of methicillin-resistant Staphylococcus aureus with an M13 repeat probe.

A bacteriophage M13 tandem repeat has been used to probe EcoRI digested genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA). The patterns generated were found to be useful in typing MRSA and generally confirmed the relationships that had previously been recognized in other studies based on antimicrobial resistance and plasmid profiles. The epidemic MRSA of London hospitals (EMRSA) and the majority of the epidemic MRSA of eastern Australian hospitals (EA MRSA) gave the same pattern. However, two isolates previously classified as EA MRSA gave a different pattern and a third another pattern. One isolate from Dublin, two isolates from Nuneaton and two isolates from Singapore gave the same pattern as the two EA MRSA. With the exception of the early or classic MRSA all the other isolates examined gave their own distinctive patterns. With one exception the classic MRSA belonged to a separate group. The exception was of particular interest because it gave the same pattern as the majority of the EA MRSA. This suggests that there may be an evolutionary relationship between some of the classic MRSA and the EMRSA of London and the EA MRSA of Australia.

Substrates respired by mitochondrial fractions of two isolates of the nematode Aphelenchus avenae and the effects of electron transport inhibitors.

Electron transport has been assayed and compared in two isolates (M and F) of the free-living (model) nematode Aphelenchus avenae. Of the substrates tested only alpha-glycerophosphate and succinate were utilised to any significant extent by both isolates. Comparative data on respiratory rates, respiratory control ratios and ADP:O ratios for various substrates are given. Succinate oxidation by isolate-F mitochondria was ca 80-90% sensitive to antimycin A while that of isolate M was almost completely refractory to antimycin A. The response to other electron transport inhibitors suggests the operation of (a) azide/cyanide sensitive, (b) azide/salicylhydroxamic acid (SHAM) insensitive but carbon monoxide sensitive and (c) SHAM-sensitive terminal oxidases to varying degrees in the mitochondria of these two isolates of A. avenae.

Study of human epithelial cell detachment and damage: effects of proteases and oxidants.

Polymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis, bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using an in vitro model of intact human amnionic epithelial cells (EC) attached to native basement membrane (BM), we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC-desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin-G (Cath-G), trypsin, and kallikrein being 3616 +/- 989 U/mL, 32.3 +/- 14.7 U/mL, 85.8 +/- 26.7 U/mL, 360 +/- 20 U/mL, 340 +/- 49 BAEE U/mL and 300 +/- 23 U/mL, respectively. Urokinase (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50 for H2O2 and HOCl being 0.59 +/- 0.006 mol/L and 0.015 +/- 0.009 mol/L, respectively) and pretreatment of EC membranes with non-detaching concentrations of H2O2 rendered them 10-fold more susceptible to protease-induced desquamation, suggesting synergism. Reduced glutathione (GSH), N-acetyl cysteine (NAC), ethylenediamine tetra-acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC-detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and alpha 1-anti-proteinase (alpha 1-AP) and, to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF, alpha 1-AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath-G induced detachment was profoundly inhibited by SBTI, GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath-G and elastase, and that PMN-mediated detachment can be prevented by Cath-G and collagenase inhibitors. The data suggest a role for proteases, particularly Cath-G and collagenase, plus oxidants in synergism with proteases, in mediating PMN-induced EC detachment.