Exploring the biophysical evidence that mammalian two pore channels are NAADP-activated calcium-permeable channels

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca2+ from acidic intracellular endo-lysosomal Ca2+-stores. It is widely accepted that two types of two pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca2+-release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca2+. Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca2+ over K+ than TPC1 and hence capable of releasing greater quantities of Ca2+ from acidic stores. TPC1 is also permeable to H+ and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca2+ release channels of the endo-lysosomal system.PostprintPeer reviewe

Modelling of Quaternary Glacier Extent and Climate in Tasmania, Australia

The aim of this study was to improve our understanding of Quaternary glaciations in Tasmania, and to assess their climatic significance. During the Quaternary, Tasmania experienced ice cap development in the West Coast Range and Central Plateau, with smaller cirque and valley glaciers developing on surrounding mountains. Geomorphic evidence indicates at least four glacial advances occurred. A 500 m resolution, three-dimensional thermomechanical ice-sheet model was used to reconstruct and simulate these glacier fluctuations. The model, while a simplification of reality, provided a framework in which to understand the genesis of contemporary landforms and former glacial climate conditions in Tasmania. Input specifications of basal topography, temperature and precipitation were required, with the latter two interpolated from present-day datasets. Numerous experiments were carried out, involving systematic alterations to temperature and precipitation, basal sliding and atmospheric temperature lapse-rates. Modelled output was compared to empirical evidence with the aim of optimizing the parameters (temperature, precipitation) from which model mismatch was minimised. An annual temperature reduction of 6' C is required for glaciers to form in Tasmania. When temperatures cool beyond this threshold, an ice mass develops over the Central Highlands, Central Plateau, West Coast Range and other mountains. In all but the most extensive glaciations, the Central Plateau ice field consists of a relatively thin carapace of ice, and the thickest and most dynamic glaciers develop in the deep valleys of the Central Highlands. The model shows that ice readily flows down the west-facing valleys from the Central Highlands to join with West Coast Range glaciers. A temperature reduction between 7 and 9' C, with corresponding regional precipitation changes of between +/-50% and an increase in orographic precipitation is required to simulate the Last Glacial Maximum (LGM) ice cover. Earlier glacial advances require larger coolings of up to 11.25' C depending on the regional precipitation conditions prescribed. Mismatches between geomorphic evidence and modelled reconstructions probably result from model grid size issues and parameters not accommodated by the model such as wind-blown snow redistribution. This means that temperature reductions derived from the model may have been overestimated. Despite these limitations, reconstructed temperatures compare well to some multi-proxy palaeo-temperature records from Tasmania, although the cooling identified was larger than that recorded in nearby ocean sediments

Disturbances of visual motion perception in bipolar disorder

OBJECTIVES: While cognitive deficits have been well documented in patients with bipolar disorder, visual perception has been less well characterized. Such deficits appear in schizophrenia, which shares genetic risk factors with bipolar disorder, and may contribute to disturbances in visual cognition and learning. METHODS: The present study investigated visual perception in bipolar disorder using psychophysical tests of contrast sensitivity, dot motion discrimination, and form discrimination. The relationship of these measures to mood state, medication status, and cognitive function was investigated. Sixty-one patients with type I bipolar disorder and 67 comparison subjects were tested. RESULTS: Results indicated a deficit in dot motion trajectory discrimination in both euthymic and ill individuals with bipolar disorder, as well as a global deficit in moving grating contrast sensitivity. Ill individuals with bipolar disorder were impaired in psychomotor processing, but this finding was not related to visual processing performance. CONCLUSIONS: These findings could be due to disturbances in specific visual pathways involved in the processing of motion properties, or to a more general deficit which impairs processing of temporally modulated stimuli

Neuropathic Nav1.3-mediated sensitization to P2X activation is regulated by protein kinase C

<p>Abstract</p> <p>Background</p> <p>Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na⁺channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.</p> <p>Results</p> <p>Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na⁺channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Na_v1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Na_v1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.</p> <p>Conclusion</p> <p>Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Na_v1.3 subunit, and the function of the Na_v1.3 channel is regulated by PKC.</p

A New Rodent Model to Assess Blood Stage Immunity to the Plasmodium falciparum Antigen Merozoite Surface Protein 119 Reveals a Protective Role for Invasion Inhibitory Antibodies

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119–specific inhibitory antibodies, but not with titres of total MSP-119–specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines

Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite.

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen

Antibodies against Merozoite Surface Protein (Msp)-119 Are a Major Component of the Invasion-Inhibitory Response in Individuals Immune to Malaria

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-119 from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-119, we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum–immune humans and P. chabaudi–immune mice. In some individual human sera, MSP-119 antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-119–based vaccines

Molecular Identification of a Malaria Merozoite Surface Sheddase

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface “sheddase,” but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite