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Neuropathic pain (NP) is a complex chronic pain state with a prevalence of almost 10% in the general population. Pharmacological options for NP are limited and slightly effective, so there is a need of developing more efficacious NP attenuating drugs. Activation of the type 1 lysophosphatidic acid (LPA(1)) receptor is a crucial factor in the initiation of NP. Hence, it is conceivable that a functional antagonism strategy could lead to NP mitigation. Here we describe a new series of LPA(1) agonists among which derivative (S)-17 (UCM-05194) stands out as the most potent and selective LPA(1) receptor agonist described so far (E(max)=118%, EC(50)=0.24 μM, K(D)=19.6 nM; inactive at autotaxin and LPA(2–6) receptors). This compound induces characteristic LPA(1)-mediated cellular effects and prompts the internalization of the receptor leading to its functional inactivation in primary sensory neurons and to an efficacious attenuation of the pain perception in an in vivo model of NP.

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in “cation-free” solution and blocked by the nonspecific Cl− channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P3−/− mice, whereas in control S1P1 floxed (S1P1fl/fl) mice and mice with a nociceptor-specific deletion of S1P1−/− receptor (SNS-S1P1−/−), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl− conductance that is largely mediated by S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain., The authors thank K. Braun, T. Martha, and M. Doblander for expert technical assistance. This work was supported by la Generalitat Valenciana and the Ministerio de Economia y Competitividad (A.V.F.M.), the Australian National Health and Medical Research Council Project Grant 535055 to R.V.H., the Intramural Research Programs of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases to R.L.P., and the Austrian Research Funding Agency FWF Project Grants P20562, P25345, and SPIN to M.K.

Summary Background Imported cases of malaria constitute an important public health problem in many countries, even in those with autochthonous cases, where disease could be acquired in these areas and then seen in non-endemic regions. Non-immune populations are susceptible to complications due to malaria infection, particularly in malaria caused by Plasmodium falciparum . However, Plasmodium vivax the predominant Plasmodium spp. in Venezuela can also lead to severe malaria. Methods We reviewed retrospectively cases of malaria to identify the clinical features of those imported cases diagnosed at two institutions in Margarita Island (a non-endemic area), Venezuela, in an 8-year period. We conducted a retrospective observational study to identify the clinical and epidemiological features among hospitalized patients at Hospital Central and Hospital Agustin Hernandez with malaria acquired at malaria-endemic locations. Results We identified eighteen imported cases of malaria confirmed by thin and thick peripheral blood smears at these two institutions over an 8-year period. The mean age of diagnosis was 27 years. P. vivax was responsible for the majority of cases. All patients presented with fever, 89% with malaise, 78% with chills, and 67% with myalgia, among others symptoms. Mean haemoglobin levels on admission were 8.1 g/dL (100% 3 (89% had platelets below 150,000); and a mean total leukocyte count: 3.4 × 10 3 cells/mm 3 (78% had leukopenia). Thirty nine percent of patients required blood transfusions. Two fatalities were identified (CFR = 11%), one associated to severe malaria due to P. falciparum and the other due to a complicated case of P. vivax malaria. Discussion Imported cases of malaria due to P. vivax and P. falciparum in the studied population are associated with significant hematological complications. These findings illustrate the importance of educating non-immune populations about the malaria risk and prevention strategies; and from a pubic health perspective, the need to develop further malaria prevention strategies at a national level.

Murine leukaemia P388 and L1210 cell sublines with varying degrees of resistance to the anthracycline daunomycin (DNM) have been used to monitor (i) intracellular accumulation of DNM, (ii) expression of the drug efflux pump P-glycoprotein (pgp) and (iii) cytoplasmic pH changes. Drug-resistant L1210/65 cells (65-fold resistance), overexpress pgp, and display decreased intracellular accumulation of DNM and identical intracellular pH as compared to the parental drug-sensitive L1210 cell line. On the other hand, moderately drug-resistant P388/20 cells (20-fold resistance), which also exhibit a decreased intracellular drug accumulation with respect to drug-sensitive P388/S cells, display only moderate pgp-encoding mdr1 gene transcription without detectable levels of pgp protein, and undergo cytoplasmic alkalinisation (up to approximately 0.2 pH units). A further increase in the level of drug resistance (P388/100 cells, 100-fold resistance), results in a more pronounced decrease in drug accumulation, significant pgp expression and slightly higher intracellular alkalinisation. Alterations in the degree of protonation of DNM have been shown previously to influence processes such as the rate of uptake and the intracellular accumulation of the drug. On this basis, we propose that the changes in intracellular pH, observed at low levels of drug resistance (P388/20 cells), could constitute an early cellular response aimed at decreasing the intracellular accumulation of ionisable anti-neoplastics. As the level of resistance increases (P388/100), the cells seem to require more efficient mechanisms of defense against the drug, such as that represented by the expression of pgp. Since there is no apparent correlation between the extent of the changes in intracellular pH and the level of pgp expression in DNM-resistant P388 cell sublines, it is suggested that these two cellular responses contributing to drug resistance could operate independently.

The scrapie prion protein (PrPsc) requires the cellular prion protein (PrPc) for its propagation and replication. In this work we studied the expression and localization of the PrPc in the central nervous system (SNC) of the rat, mouse, cat, cow and human, using immunohistochemistry and Western blot techniques to understand more about prionopathies and Alzheimer's disease (EA).For the immunohistochemistry study we used human, cat, rat and cow samples to analyse frontal, temporal and occipital cortex, as well as the hippocampus and the thalamus. For the Western blot analysis we used mouse, cat, cow and human brain samples.We observed a decrease in the amount of PrPc in the SNC in a rostrocaudal shift in the species mentioned above. We observed inhibitory cells in the cat cortex. The Western blot analysis showed a similar pattern of expression in the different species studied with a preponderance of the diglycosylated band, in relation to the other bands observed in the analysis.These data suggest that in prionopathies PrPsc could be transmitted and could be replicated in and from the areas with most expression of PrPc. Similarly, a higher amount of this protein (PrPc) in some brain areas could explain some histopathological aspects of EA.Our findings support the hypothesis of a retrograde transport of PrPsc in the SNC. PrPc could be related to the pathophysiology of EA.

When the anthracycline daunomycin (DNM) is incorporated into isolated plasma membranes from P388 murine leukemia cells, the drug partitions between ‘deep’ and ‘surface’ membrane domains. Such domains have been characterized on the basis of: (1) fluorescence resonance energy transfer between 1,6-diphenylhexa-1,3,5-triene or 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene as energy donors, which are well known in their positioning within the membrane, and daunomycin as the energy acceptor, and (2) quenching of the fluorescence of the membrane-associated drug by the water-soluble quencher iodide. The distribution of DNM between the two plasma membrane domains is different depending on the cellular phenotype. Thus, in membranes from drug-sensitive cells, DNM is preferentially confined to ‘surface’ domains, while in membranes from drug-resistant cells, the drug distributes more homogeneously between ‘surface’ and ‘deep’ domains. Experiments using artificial lipid vesicles suggest that differences in the relative levels of certain lipids in the plasma membranes from drug-sensitive and drug-resistant cells, namely phosphatidylserine and cholesterol, are partly responsible for the observed differences in the distribution of DNM. Since drug-membrane interactions are important in anthracycline cytotoxicity, it is possible that our observations on a different membrane distribution of daunomycin, may be related to the different sensitivity to the drug exhibited by these cells., This work has been partly supported by Grants PB87-07qO (to J.M.G.-R.) and PB87-0791 (to J.A.F.) from the DGICT of Spain.

Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.

Waste stabilization ponds (WSP) are an often-used option to treat faecal sludges collected from on-site sanitation systems. Since agricultural use is one of the most attractive options for sludge disposal, specific guidelines on the hygienic sludge quality must be fulfilled, such as for viable helminth eggs and Salmonella sp. Although Salmonella isolation methods are well known for other types of samples, they are not suitable for faecal sludge. The reason can be attributed to the co-existence of a native bacterial sludge flora masking Salmonella development, especially if this bacteria is present at low concentrations. In order to select the best methodology for Salmonella recovery from septage sludge, different culture media were assayed at different incubation periods and temperatures. The proposed methodology for Salmonella recovery from sludge can be summarised as follows: (1) enrichment in Rappaport-Vassiliadis broth at 43 degrees C, 48 hours, and (2) isolation in XLD agar at 40 degrees C, 24 hours. Identification of suspected colonies by biochemical tests: TSI, LIA, urease and serological confirmation with Group O Antigen.

Sludge management arises as a relevant problem after being accumulated in primary ponds of septage treatment plants. One of the most attractive options for sludge disposal is its use in agriculture and then specific guidelines regarding hygienic quality must be fulfilled. This study aimed at evaluating the storage time needed to inactivate Ascaris eggs and Salmonella in sludge accumulated in a primary pond treating septage. Raw septage exhibited very low concentrations of viable Ascaris eggs, thus experiments with Ascaris suum eggs spiking were conducted. The concentration of Ascaris eggs in the solids accumulated at the bottom of the pond was 20 eggs/g of total solids (g TS) at the time of pond closure. Although it decreased, some eggs remained viable (0.59 mean viable eggs/g TS) up to 20 months of in-pond storage of the biosolids. Salmonella survival was studied after developing an analytical method that inhibited the native flora. Sludge was seeded with Salmonella enteritidis. An equation adequately describing Salmonella die-off in biosolids subjected to 115 days of in-pond storage/dewatering, was found to be represented by the regression: y = log MPN Salmonella/g TS = 6.67 x t(-0.086), with t = storage time elapsed in days. The initial concentration was 7.0 x 10(6) MPN/g TS and the removal efficiency was 99%.

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [(3) H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [(3)H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [(3)H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release, presumably by cleaving SNAP-25, a protein involved in docking of synaptic vesicles with the presynaptic plasma membrane. Three excitation-secretion uncoupling peptides (ESUPs), which mimic the carboxy-terminal domain of SNAP-25 and span or adjoin the cleavage sites for BoNT/A and BoNT/E, also inhibit transmitter release from permeabilized bovine chromaffin cells. In this study, these peptides were tested for effects on acetylcholine (ACh) release at an identified cholinergic synapse in isolated buccal ganglia of Aplysia californica. The presynaptic neuron was stimulated electrically to elicit action potentials. The postsynaptic neuron was voltage-clamped, and evoked inhibitory postsynaptic currents (IPSCs) were recorded. The ESUPs were pressure-injected into the presynaptic neuron, and their effects on the amplitude of the IPSCs were studied. Acetylcholine release from presynaptic cells, as measured by IPSC amplitudes, was gradually inhibited by the ESUPs. All three peptides caused ca. 40% reduction in IPSC amplitude in 2 h. Random-sequence peptides of the same amino acid composition had no effect. Injection of BoNT/E, in contrast, caused ca. 50% reduction in IPSC amplitude in 30 min and almost complete inhibition in 2 h. These results are the first demonstration that ESUPs block neuronal cholinergic synaptic transmission. They are consistent with the concept that ESUPs compete with the intact SNAP-25 for binding with other fusion proteins, thus inhibiting stimulus-evoked exocytosis of neurotransmitter.

The effect of several aliphatic aldehydes on lipid peroxidation was evaluated by measuring the oxygen uptake rate, thiobarbituric acid-reactive products formation and the emitted visible chemiluminescence intensity. Measurements were carried out in brain homogenates and erythrocyte plasma membrane and liver microsomal fractions. In all systems studied, aldehydes (25 mmol/L) (e.g. acetaldehyde, 2,2-dimethylpropanal), increased the intensity of the luminescence associated with the oxidation process. In contrast, aldehyde incorporation decreased TBARS production and the rate of oxygen uptake. The increased luminescence intensity is explained in terms of secondary reactions of aldehyde derived free radicals. These results clearly indicate that extreme care must be exercised in the interpretation of chemiluminescence data in the presence of aldehydes.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.

The effect of age on the glutathione antioxidant system and its acinar distribution in rat liver was studied. GSH/GSSG ratio in blood and liver was lower in old than in young rats. Hepatic glutathione peroxidase and glutathione S-transferase activities were higher in old than in young rats, whereas hepatic gamma-glutamyl transpeptidase activity was lower in old than in young rats. Glutathione reductase and glucose-6-phosphate dehydrogenase activities did not change with age in rat liver. Total glutathione levels and glutathione peroxidase activity were higher in periportal than in perivenous areas of young rats, but this heterogeneous distribution did not occur in old rats. No change with age was found in hepatic zonation of glutathione reductase and glucose-6-phosphate dehydrogenase.

Eye lenses from young rats or mice synthesize GSH from methionine or N-acetylcysteine. However, lenses from old animals do not synthesize GSH from methionine. This is due to the absence of cystathionase activity in old lenses. GSH monoethyl ester, but not free GSH, increases GSH content and protects the lens against experimental oxidative stress. The importance of these results in the prevention of cataractogenesis is discussed.

1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257–1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333–7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane ‘surface’ domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927–3932), thus acting as a drug ‘sink’. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change inKm. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased theVmaxof DA transport without alteringKm. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

The cellular prion protein (PrP(C)) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrP(Sc,) also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrP(C) is needed for the establishment and further evolution of prions. The present work compares the expression and localization of PrP(C) between healthy human brains and those suffering from Alzheimer disease (AD). In both situations we have observed a rostrocaudal decrease in the amount of PrP(C) within the CNS, both by immunoblotting and immunohistochemistry techniques. PrP(C) is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrP(C) in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands. With regards to amyloid plaques, those present in AD cases were positively labeled for PrP(C), a result which is further supported by the presence of PrP(C) in the amyloid plaques of a transgenic line of mice mimicking AD. The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.

changes cannot follow from changes in fluidity. More recently, we have shown that nonylphenol, which strongly inhibits ATPase activity, reduces the equilibrium level of phosphorylation of the ATPase by phosphate, again a result which cannot be explained in terms of effects of fluidity (F. Michelangeli, S. Orlowski, P. Champed, J. M. East & A. G. Lee, unpublished work). The idea that the activity of membrane-bound enzymes should be sensitive to membrane fluidity is intrinsically attractive for, after all, we know that enzymes undergo conformational changes and it seems reasonable that the more fluid the environment the easier will be these'changes. Nevertheless, for the (Ca"-Mg'+)-ATPase there is no evidence that the fluidity of the surrounding lipid has any significant effect on activity, while there is evidence that other effects must be important. There seems to be no reason to suppose that the ATPase is in any way unusual in this respect.