Dlgh1 and Carma1 MAGUK proteins contribute to signal specificity downstream of TCR activation.

Mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), have a key role in T cell receptor (TCR)-induced gene transcription but their precise mechanism of activation is not well understood. The findings of two recent papers provide new insight into the activation of p38 and JNK by the membrane-associated guanylate kinase (MAGUK) family members Dlgh1 and Carma1, respectively, and show how distinct MAGUK proteins control specific aspects of TCR-mediated MAPK activation.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity.

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Effects of alternative sets of climatic predictors on species distribution models and associated estimates of extinction risk: a test with plants in an arid environment

Aim To evaluate the effects of using distinct alternative sets of climatic predictor variables on the performance, spatial predictions and future projections of species distribution models (SDMs) for rare plants in an arid environment. . Location Atacama and Peruvian Deserts, South America (18º30'S - 31º30'S, 0 - 3 000 m) Methods We modelled the present and future potential distributions of 13 species of Heliotropium sect. Cochranea, a plant group with a centre of diversity in the Atacama Desert. We developed and applied a sequential procedure, starting from climate monthly variables, to derive six alternative sets of climatic predictor variables. We used them to fit models with eight modelling techniques within an ensemble forecasting framework, and derived climate change projections for each of them. We evaluated the effects of using these alternative sets of predictor variables on performance, spatial predictions and projections of SDMs using Generalised Linear Mixed Models (GLMM). Results The use of distinct sets of climatic predictor variables did not have a significant effect on overall metrics of model performance, but had significant effects on present and future spatial predictions. Main conclusion Using different sets of climatic predictors can yield the same model fits but different spatial predictions of current and future species distributions. This represents a new form of uncertainty in model-based estimates of extinction risk that may need to be better acknowledged and quantified in future SDM studies.

Lack of MRI neurohypophyseal bright signal in a child with congenital nephrogenic diabetes insipidus

Congenital nephrogenic diabetes insipidus (CNDI) is a rare disease characterized by the inability of the kidney to respond to arginine vasopressin (AVP). The absence of the neurohypophyseal 'bright signal' on T1 sequence magnetic resonance imaging (MRI) is considered as an argument in favour of the diagnosis of central diabetes insipidus (CDI). This observation is challenged as we hereby present a case of a child diagnosed with CNDI and who did not present MRI pituitary bright signal. A 6-month-old male presented with failure to thrive, polyuria and polydypsia. Family history revealed that the mother, 35 years of age, had been presenting polydypsia and polyuria, and she was investigated at the age of 6 years with no concluding diagnosis. The patient's physical exam showed a weight of 5215 g (−3 DS) and clinical signs of dehydration. The patient's plasma sodium level was 155 mmol/L, osmolality 305 mOsm/kg and urine osmolality 150 mOsm/kg. Brain MRI showed in T1 sequences the absence of the posterior pituitary bright signal suggesting the diagnosis of CDI (Figure 1). The child was treated with synthetic AVP analogue 1-desamino-8-d-arginine vasopressin (DDAVP) without improvement, which led to the consideration of CNDI. The diagnosis was confirmed by an elevated serum level of AVP of 214 pmol/L (reference value ≤4.34 pmol/L) and by genetic analysis demonstrating and T106C mutation in the V2R (X-linked CNDI). The child was treated with thiazide diuretic and increased fluids with restricted sodium intake. This resulted in catch-up growth and improved neurological development. A follow-up MRI was performed 6 months after the start of therapy with the same technique. At that time, the child's weight had improved to 9310 g (−1.5 DS) corresponding to a gain of 22 g per day, and he did not present any clinical signs of dehydration and had a normal plasma level of sodium (140 mmol/L). MRI showed that the bright signal was still absent.

Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family.

Jasmonic acid and its precursors are potent regulatory molecules in plants. We devised a method for the simultaneous extraction of these compounds from plant leaves to quantitate changes in the levels of jasmonate family members during health and on wounding. During our study, we identified a novel 16-carbon cyclopentenoic acid in leaf extracts from Arabidopsis and potato. The new compound, a member of the jasmonate family of signals, was named dinor-oxo-phytodienoic acid. Dinor-oxo-phytodienoic acid was not detected in the Arabidopsis mutant fad5, which is incapable of synthesizing 7Z,10Z, 13Z-hexadecatrienoic acid (16:3), suggesting that the metabolite is derived directly from plastid 16:3 rather than by beta-oxidation of the 18-carbon 12-oxo-phytodienoic acid. Simultaneous quantitation of jasmonate family members in healthy leaves of Arabidopsis and potato suggest that different plant species have different relative levels of jasmonic acid, oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. We term these profiles "oxylipin signatures." Dinor-oxo-phytodienoic acid levels increased dramatically in Arabidopsis and potato leaves on wounding, suggesting roles in wound signaling. Treatment of Arabidopsis with micromolar levels of dinor-oxo-phytodienoic acid increased the ability of leaf extracts to transform linoleic acid into the alpha-ketol 13-hydroxy-12-oxo-9(Z) octadecenoic acid indicating that the compound can regulate part of its own biosynthetic pathway. Tightly regulated changes in the relative levels of biologically active jasmonates may permit sensitive control over metabolic, developmental, and defensive processes in plants.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Malarial hemozoin is a Nalp3 inflammasome activating danger signal.

BACKGROUND: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. METHODOLOGY/PRINCIPAL FINDINGS: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. SIGNIFICANCE/CONCLUSIONS: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Is the macromolecule signal tissue-specific in healthy human brain? A (1)H MRS study at 7 Tesla in the occipital lobe.

PURPOSE: The macromolecule signal plays a key role in the precision and the accuracy of the metabolite quantification in short-TE (1) H MR spectroscopy. Macromolecules have been reported at 1.5 Tesla (T) to depend on the cerebral studied region and to be age specific. As metabolite concentrations vary locally, information about the profile of the macromolecule signal in different tissues may be of crucial importance. METHODS: The aim of this study was to investigate, at 7T for healthy subjects, the neurochemical profile differences provided by macromolecule signal measured in two different tissues in the occipital lobe, predominantly composed of white matter tissue or of grey matter tissue. RESULTS: White matter-rich macromolecule signal was relatively lower than the gray matter-rich macromolecule signal from 1.5 to 1.8 ppm and from 2.3 to 2.5 ppm with mean difference over these regions of 7% and 12% (relative to the reference peak at 0.9 ppm), respectively. The neurochemical profiles, when using either of the two macromolecule signals, were similar for 11 reliably quantified metabolites (CRLB < 20%) with relatively small concentration differences (< 0.3 μmol/g), except Glu (± 0.8 μmol/g). CONCLUSION: Given the small quantification differences, we conclude that a general macromolecule baseline provides a sufficiently accurate neurochemical profile in occipital lobe at 7T in healthy human brain.

Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

Signal transduction in plant-beneficial rhizobacteria with biocontrol properties.

Biological control of root pathogens--mostly fungi--can be achieved by the introduction of selected bacterial inoculants acting as 'biopesticides'. Successful inoculants have been identified among Gram-negative and Gram-positive bacteria, often belonging to Pseudomonas spp. and Bacillus spp., respectively. Biocontrol activity of a model rhizobacterium, P. fluorescens CHAO, depends to a considerable extent on the synthesis of extracellular antimicrobial secondary metabolites and exoenzymes, thought to antagonize the pathogenicity of a variety of phytopathogenic fungi. The regulation of exoproduct formation in P. fluorescens (as well as in other bacteria) depends essentially on the GacS/GacA two-component system, which activates a largely unknown signal transduction pathway. However, recent evidence indicates that GacS/GacA control has a major impact on target gene expression at a post-transcriptional level, involving an mRNA target sequence (typically near the ribosome binding site), two RNA binding proteins (designated RsmA and RsmE), and a regulatory RNA (RsmZ) capable of binding RsmA. The expression and activity of the regulatory system is stimulated by at least one low-molecular-weight signal. The timing and specificity of this switch from primary to secondary metabolism are essential for effective biocontrol.

Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia.

Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens.

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.