mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.     

Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons

Activation of P2X receptors by a Ca²⁺- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca²⁺ ionophore ionomycin (1.5 and 3 µM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca²⁺, depolarizing voltage steps (–60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba²⁺ for Ca²⁺ augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca²⁺] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca²⁺ with Mg²⁺, demonstrating a Ca²⁺ requirement. ASTICs were Na⁺ dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 µM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 µM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 µM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors. sympathetic; purinergic; neurotransmission; phorbol ester; botulinum toxin     

Fuzzy subset approach in coupled population dynamics of blowflies

This paper is a study on the population dynamics of blowflies employing a density-dependent, non-linear mathematical model and a coupled population formalism. In this study, we investigated the coupled population dynamics applying fuzzy subsets to model the population trajectory, analyzing demographic parameters such as fecundity, survival, and migration. The main results suggest different possibilities in terms of dynamic behavior produced by migration in coupled populations between distinct environments and the rescue effect generated by the connection between populations. It was possible to conclude that environmental heterogeneity can play an important role in blowfly metapopulation systems. The implications of these results for population dynamics of blowflies are discussed.     

Interaction of PDZRhoGEF with microtubule-associated protein 1 light chains: link between microtubules, actin cytoskeleton, and neuronal polarity

Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, we screened for proteins that interact with the N terminus of rPDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). We report here that rPDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rPDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity.     

Benthic decomposition of Ulva lactuca: A controlled laboratory experiment

The degradation of an Ulva lactuca mat (0.2 kg dw m⁻²) was studied in a controlled flow-through mesocosm for 31 d. Sediment chambers without U. lactuca served as controls. Fluxes of ∑CO₂, O₂, inorganic nitrogen, and urea were determined during the incubation period in addition to sulfate reduction rates, POC and PON content, enumeration of specific bacterial populations and evaluation of the physiological state of the added U. lactuca thalli. After U. lactuca addition to the chambers, there was an immediate increase in the efflux of ∑CO₂ from 11 to 27 mmol-C m⁻² d⁻¹ and a concomitant increase in O₂ uptake from 11 to 23 mmol m⁻² d⁻¹. These effluxes remained elevated throughout the incubation period. In contrast, the NH₄⁺ efflux increased from 0.1 to 1.8 mmol NH₄⁺ m⁻² d⁻¹ during the first 3 d of incubation, followed by 6 d with a constant efflux rate, after which time it decreased gradually to 0.3 mmol NH₄⁺ m⁻² d⁻¹ by the end of the experiment. In total, NH₄⁺accounted for 83% of the total nitrogen efflux after addition of U. lactuca. During the 31 d incubation period there was a continuous colonization of the thalli by bacteria. Sulfate reducers associated with the thalli accounted for 3% of the carbon oxidation on day 31. The molar C:N ratio in mineralization products (the ratio between the efflux of ∑CO₂ and NH₄⁺ + NO₂⁻ + NO₃⁻) increased from 15 mol mol⁻¹ at day 11 after U. lactuca addition to >80 mol mol⁻¹ by the end of the incubation. Since the C:N ratio in the mineralization products was much higher than the original thallus material (8.9 mol mol⁻¹) it is probable that a preferential incorporation of NH₄⁺ into the increasing bacterial biomass occurred. The nitrogen for bacterial growth was most likely obtained from degradation of U. lactuca thalli as there was no stimulation of urea-N turnover in the sediment during incubation. The net increase in bacteria cell number in the 18-mm thick thallus layer was estimated to be 7.6 × 10⁹ to 2.4 × 10¹⁰ bacterial cells cm⁻³. In contrast, the bacterial cell number remained constant in the −Ulva incubations.     

Cytotoxic activity of Bacillus anthracis protective antigen observed in a macrophage cell line overexpressing ANTXR1

Anthrax toxin protective antigen (PA) binds cell surface receptors (e.g. ANTXR1,2), forms heptameric pores, and translocates lethal factor (LF) or oedema factor (OF) into the cytoplasm of mammalian cells. In the current study, we sought to determine how receptor levels influence these events, by examining PA heptamer stability and related processes in macrophages that overexpress ANTXR1 (RAW 264.7ANTXR1). In these experiments, PA-oligomers demonstrated an extended half-life in RAW 264.7ANTXR1 macrophages, with SDS-resistant heptamers detected up to 10 h following treatment, while levels of PA-oligomers declined within 3 h in control cells. RAW 264.7ANTXR1 macrophages were also more sensitive to lethal toxin, a combination of PA and LF. Surprisingly, we found that PA alone was cytotoxic to RAW 264.7ANTXR1 cells. Further analysis found that PA cytotoxicity required direct interaction with ANTXR1, oligomerization, channel formation, endosomal acidification, and was independent of the ANTXR1 cytoplasmic tail. PA intoxication of RAW 264.7ANTXR1 macrophages resulted in caspase-3 activation, with corresponding DNA fragmentation and proteolytic cleavage of poly-ADP-ribose polymerase, as well as activation of Bid, suggesting cell death occurred via apoptosis. Overall, results from the current study suggest that receptor levels dictate the extent of PA oligomer stability, and shifts in this normal process can lead to cell death via apoptosis in the absence of toxin catalytic subunits.     

Population genetic estimation of the loss of genetic diversity during horizontal transmission of HIV-1

Background  

Genetic diversity of the human immunodeficiency virus type 1 (HIV-1) population within an individual is lost during transmission to a new host. The demography of transmission is an important determinant of evolutionary dynamics, particularly the relative impact of natural selection and genetic drift immediately following HIV-1 infection. Despite this, the magnitude of this population bottleneck is unclear.     

Literacy and recent history of diarrhoea are predictive of Plasmodium falciparum parasitaemia in Kenyan adults

Background

Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked.

Methods

The^ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses.

Results

Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa.

Conclusion

The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.