Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.     

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.     

Homologies among different adductor mandibulae sections of teleostean fishes, with special regard to catfishes (Teleostei: siluriformes)

The adductor mandibulae complex has been a subject of discussion and uncertainties due to a wide range of differentiations and fusions that have occurred during teleost evolution. The adductor mandibulae of numerous catfishes was studied in detail and compared with that of several other teleosts described in the literature. Our observations and comparisons demonstrate that: 1) the adductors mandibulae Aomega, A2, and A3 of acanthopterygians correspond, respectively, to the Aomega, A2, and A3 of ostariophysines; 2) the antero-dorso-lateral (A1) and the antero-ventro-lateral (A1-OST) sections of the adductor mandibulae present, respectively, in acanthopterygians and in basal ostariophysines are the result of two different patterns of differentiation of this muscle; 3) some derived ostariophysines present a lateral section of the adductor mandibulae attached to the upper jaw (A0) that is not homologous with any other section of this muscle present in any other ostariophysine or acanthopterygian fish; 4) the configuration of the adductor mandibulae present in Diplomystes seems to be the plesiomorphic condition for catfishes; and 5) the muscle retractor tentaculi, present in a large number of catfishes, is derived from the inner section of the adductor mandibulae (A3) and, thus, is not homologous with the lateral bundle of this muscle (A0) that inserts on the upper jaw in some derived ostariophysine fishes.     

Visual processing: the devil is in the details

Ganglion cells convey information from the retina back to the brain. Recent experiments have examined how ganglion cell receptive fields are assembled from many incoming signals.     

Long-Lasting Ibogaine Protection against NMDA-Induced Convulsions in Mice

Ibogaine, a putative antiaddictive drug, is remarkable in its apparent ability to downgrade withdrawal symptoms and drug craving for extended periods of time after a single dose. Ibogaine acts as a non-competitive NMDA receptor antagonist, while NMDA has been implicated in long lasting changes in neuronal function and in the physiological basis of drug addiction. The purpose of this study was to verify if persistent changes in NMDA receptors could be shown in vivo and in vitro after a single administration of ibogaine. The time course of ibogaine effects were examined on NMDA-induced seizures and [³H] MK-801 binding to cortical membranes in mice 30min, 24, 48, and 72h post treatment. Ibogaine (80 mg/kg, ip) was effective in inhibiting convulsions induced by NMDA at 24 and 72 hours post administration. Likewise, [³H] MK-801 binding was significantly decreased at 24 and 72 h post ibogaine. No significant differences from controls were found at 30min or 48h post ibogaine. This long lasting and complex pattern of modulation of NMDA receptors prompted by a single dose of ibogaine may be associated to its antiaddictive properties.     

Compounds Extracted from Phyllantus and Jatropha elliptica Inhibit the Binding of [3H]Glutamate and [3H]GMP-PNP in Rat Cerebral Cortex Membrane

Glutamate is to be considered a nociceptive neurotransmitter and glutamatergic antagonists present antinoceptive activity. In this study we investigated the effects of the naturally occurring antinociceptive compounds rutin, geraniin and quercetine extracted from Phyllanthus, as well as the diterpene jatrophone, extracted from Jatropha elliptica on the binding of [³H]glutamate and [³H]GMP-PNP [a GTP analogue which binds to extracellular site(s), modulating the glutamatergic transmission] in rat brain membrane. Jatrophone inhibited [³H]glutamate binding and geraniin inhibited [³H]GMP-PNP binding. Quercetine inhibited the binding of both ligands. These results may indicate a neurochemical parameter possibly related to the antinoceptive activity of these natural compounds.     

Isolation and characterization of an equine foamy virus

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.     

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with ¹²⁵I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.     

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.     

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.