Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons.

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

Kalemia during combined therapy with an angiotensin converting enzyme inhibitor and a potassium-sparing diuretic.

Both angiotensin converting enzyme (ACE) inhibitors and potassium-sparing diuretics tend to increase serum potassium levels. This retrospective study was undertaken to assess whether these two types of agents can nevertheless be combined safely. Twelve hypertensive patients were treated for 1-70 months (mean = 17) with an ACE inhibitor together with a potassium-sparing diuretic (spironolactone, n = 10; amiloride, n = 2). In addition, eight patients also took a thiazide or a loop diuretic. Nine patients had a normal and three a slightly impaired renal function. No clinically relevant hyperkalemia was observed during the course of the study. These data suggest that it is not impossible to combine an ACE inhibitor with a potassium-sparing diuretic, as long as renal function is normal and serum potassium concentration is monitored closely.

Role of bradykinin in the neonatal renal effects of angiotensin converting enzyme inhibition.

The vascular effects of angiotensin converting enzyme inhibitors are mediated by the inhibition of the dual action of angiotensin converting enzyme (ACE): production of angiotensin II and degradation of bradykinin. The deleterious effect of converting enzyme inhibitors (CEI) on neonatal renal function have been ascribed to the elevated activity of the renin-angiotensin system. In order to clarify the role of bradykinin in the CEI-induced renal dysfunction of the newborn, the effect of perindoprilat was investigated in anesthetized newborn rabbits with intact or inhibited bradykinin B2 receptors. Inulin and PAH clearances were used as indices of GFR and renal plasma flow, respectively. Perindoprilat (20 microg/kg i.v.) caused marked systemic and renal vasodilation, reflected by a fall in blood pressure and renal vascular resistance. GFR decreased, while urine flow rate did not change. Prior inhibition of the B2 receptors by Hoe 140 (300 microg/kg s.c.) did not prevent any of the hemodynamic changes caused by perindoprilat, indicating that bradykinin accumulation does not contribute to the CEI-induced neonatal renal effects. A control group receiving only Hoe 140 revealed that BK maintains postglomerular vasodilation via B2 receptors in basal conditions. Thus, the absence of functional B2 receptors in the newborn was not responsible for the failure of Hoe 140 to prevent the perindoprilat-induced changes. Species- and/or age-related differences in the kinin-metabolism could explain these results, suggesting that in the newborn rabbit other kininases than ACE are mainly responsible for the degradation of bradykinin.

NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non-coding RNA: NrsZ (nitrogen-regulated sRNA). We show that under nitrogen limitation, NrsZ is induced by the NtrB/C two component system, an important regulator of nitrogen assimilation and P. aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN. Furthermore, we demonstrate that NrsZ modulates P. aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post-transcriptional control of rhlA, a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions.

Temperature-dependent elasticity of microtubules

Central to the biological function of microtubules is their ability to modify their length which occurs by addition and removal of subunits at the ends of the polymer, both in vivo and in vitro. This dynamic behavior is strongly influenced by temperature. Here, we show that the lateral interaction between tubulin subunits forming microtubule is strongly temperature dependent. Microtubules deposited on prefabricated substrates were deformed in an atomic force microscope during imaging, in two different experimental geometries. Microtubules were modeled as anisotropic, with the Young's modulus corresponding to the resistance of protofilaments to stretching and the shear modulus describing the weak interaction between the protofilaments. Measurements involving radial compression of microtubules deposited on flat mica confirm that microtubule elasticity depends on the temperature. Bending measurements performed on microtubules deposited on lithographically fabricated substrates show that this temperature dependence is due to changing shear modulus, implying that the lateral interaction between the protofilaments is strongly determined by the temperature. These measurements are in good agreement with previously reported measurements of the disassembly rate of microtubules, demonstrating that the mechanical and dynamic properties of microtubules are closely related.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

Jasmonate controls polypeptide patterning in undamaged tissue in wounded Arabidopsis leaves.

Wounding initiates a strong and largely jasmonate-dependent remodelling of the transcriptome in the leaf blades of Arabidopsis (Arabidopsis thaliana). How much control do jasmonates exert on wound-induced protein repatterning in leaves? Replicated shotgun proteomic analyses of 2.5-mm-wide leaf strips adjacent to wounds revealed 106 differentially regulated proteins. Many of these gene products have not emerged as being wound regulated in transcriptomic studies. From experiments using the jasmonic acid (JA)-deficient allene oxide synthase mutant we estimated that approximately 95% of wound-stimulated changes in protein levels were deregulated in the absence of JA. The levels of two tonoplast proteins already implicated in defense response regulation, TWO-PORE CHANNEL1 and the calcium-V-ATPase ACA4 increased on wounding, but their transcripts were not wound inducible. The data suggest new roles for jasmonate in controlling the levels of calcium-regulated pumps and transporters, proteins involved in targeted proteolysis, a putative bacterial virulence factor target, a light-dependent catalyst, and a key redox-controlled enzyme in glutathione synthesis. Extending the latter observation we found that wounding increased the proportion of oxidized glutathione in leaves, but only in plants able to synthesize JA. The oxidizing conditions generated through JA signaling near wounds help to define the cellular environment in which proteome remodelling occurs.

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Genetic structure of the genus Leptospira by multilocus enzyme electrophoresis

Thirty strains from the 11 species of the genus Leptospira were studied by multilocus enzyme electrophoresis at 12 enzyme loci, all of which were polymorphic. The mean number of alleles per locus was 6.5. Twenty-five electrophoretic types were distinguished. Grouping of the strains by cluster analysis was in general agreement with species delineation as determined by DNA-DNA hybridization, except for the strains of Leptospira meyeri and Leptospira inadai, which were scattered throughout the genus, reflecting previously recognized taxonomic uncertainties. Analysis of the clonality within Leptospira interrogans sensu stricto indicated that this population was relatively heterogeneous and a lack of gene linkage disequilibrium could not be excluded. There was a genetic discrimination between the pathogenic species and the saprophytic ones. The phenotypically intermediate species (L. inadai and Leptospira fainei) were also genetically separated and were probably closer to the saprophytes than to the pathogens.

Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

Morphological and physiological species-dependent characteristics of the rodent Grueneberg ganglion.

In the mouse, the Grueneberg ganglion (GG) is an olfactory subsystem implicated both in chemo- and thermo-sensing. It is specifically involved in the recognition of volatile danger cues such as alarm pheromones and structurally-related predator scents. No evidence for these GG sensory functions has been reported yet in other rodent species. In this study, we used a combination of histological and physiological techniques to verify the presence of a GG and investigate its function in the rat, hamster, and gerbil comparing with the mouse. By scanning electron microscopy (SEM) and transmitted electron microscopy (TEM), we found isolated or groups of large GG cells of different shapes that in spite of their gross anatomical similarities, display important structural differences between species. We performed a comparative and morphological study focusing on the conserved olfactory features of these cells. We found fine ciliary processes, mostly wrapped in ensheating glial cells, in variable number of clusters deeply invaginated in the neuronal soma. Interestingly, the glial wrapping, the amount of microtubules and their distribution in the ciliary processes were different between rodents. Using immunohistochemistry, we were able to detect the expression of known GG proteins, such as the membrane guanylyl cyclase G and the cyclic nucleotide-gated channel A3. Both the expression and the subcellular localization of these signaling proteins were found to be species-dependent. Calcium imaging experiments on acute tissue slice preparations from rodent GG demonstrated that the chemo- and thermo-evoked neuronal responses were different between species. Thus, GG neurons from mice and rats displayed both chemo- and thermo-sensing, while hamsters and gerbils showed profound differences in their sensitivities. We suggest that the integrative comparison between the structural morphologies, the sensory properties, and the ethological contexts supports species-dependent GG features prompted by the environmental pressure.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes.

A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl-transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH-cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes.

Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.