Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation.     

European porphyria initiative (EPI): a platform to develop a common approach to the management of porphyrias and to promote research in the field

Porphyrias are uncommon inherited diseases of haem biosynthesis for which the diagnosis and treatment varies in individual countries. Despite the existence of guidelines recommended by porphyria experts concerning the diagnosis and management of the acute porphyrias, and of specialist centres in most European countries, many clinicians still do not apply these guidelines. The European Porphyia Initiative (EPI) network was formed in 2001 in order to compare experience among countries to attempt to develop a common approach to the management of the porphyrias, particularly concerning recommendation of safe and unsafe drugs, and to facilitate international collaborative clinical and biological research. The main achievements of EPI during this period have been: * Drafting and agreeing to consensus protocols for the diagnosis and management of acute hepatic porphyrias. * Creation of a multilingual website, particularly focusing on guidelines for common prescribing problems in acute porphyria and on providing information for patients that is now available in 10 languages: (www.porphyria-europe.org). EPI's current objectives are to develop the EPI platform, expand to new countries, extend to non-acute porphyrias and design European research and clinical trials in porphyria. The project will focus on: 1. Setting up a European laboratory external quality assurance scheme (EQAS) for biochemical and molecular investigations and their interpretation 2. Establishing a consensus drug list in collaboration with the Nordic porphyria network 3. Improving patient counseling 4. Developing large multi-centre, multi-national research projects. Due to the rarity of the porphyrias, it would be very difficult for any one country to provide this data with a sufficient number of patients and within a reasonable timescale. The progress achieved will facilitate improvements in the treatment and development of new therapeutic strategies. It will set a pattern for establishing, and subsequently harmonising, between countries best clinical practice for a rare but important group of diseases, and will help to develop the optimal therapy and ensure its cost effectiveness.     

Novel method for high-throughput colony PCR screening in nanoliter-reactors

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr^+/+ and cftr^−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.     

Insecticidal peptides from the theraposid spider Brachypelma albiceps: An NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.     

Calcium Is a Major Determinant of Xylem Vulnerability to Cavitation

Xylem vulnerability to cavitation is a key parameter in the drought tolerance of trees, but little is known about the control mechanisms involved. Cavitation is thought to occur when an air bubble penetrates through a pit wall, and would hence be influenced by the wall''s porosity. We first tested the role of wall-bound calcium in vulnerability to cavitation in Fagus sylvatica. Stems perfused with solutions of oxalic acid, EGTA, or sodium phosphate (NaPO₄) were found to be more vulnerable to cavitation. The NaPO₄-induced increase in vulnerability to cavitation was linked to calcium removal from the wall. In contrast, xylem hydraulic conductance was unaffected by the chemical treatments, demonstrating that the mechanisms controlling vulnerability to cavitation and hydraulic resistance are uncoupled. The NaPO₄ solution was then perfused into stems from 13 tree species possessing highly contrasted vulnerability to cavitation. Calcium was found to be a major determinant of between-species differences in vulnerability to cavitation. This was evidenced in angiosperms as well as conifer species, thus supporting the hypothesis of a common mechanism in drought-induced cavitation.In plants, long-distance sap transport occurs under negative pressures in xylem conduits. Sap flows between adjoining conduits through pits that form pores in the walls, and that facilitate the flow of water while preventing the passage of air bubbles. Under water stress conditions, xylem tensions increase and the conduits become vulnerable to cavitation. Cavitation provokes an air embolism that leads to a loss of hydraulic conductance, thus exacerbating plant water deficit.Species resistance to cavitation has been intensively studied over the past two decades, and is now ranked among the traits with the highest functional and ecological significance. In woody species for instance, xylem vulnerability to cavitation correlates tightly with species-specific drought tolerance (Pockman and Sperry, 2000; Tyree et al., 2003; Maherali et al., 2004), with more xerophilous species proving less vulnerable to cavitation. Substantial variations have also been found between genotypes of a different species (Cochard et al., 2007; Dalla-Salda et al., 2009). This implies that this trait could potentially be used in breeding programs to identify more drought-tolerant species or genotypes. However, efforts in this direction are still strongly impeded by a lack of understanding of the molecular and genetic basis of cavitation resistance. Our work represents a first significant step toward resolving this challenging issue.Understanding the fine mechanism of cavitation formation is a pivotal step toward identifying the key structures and the key genes coding for these structures, yet we currently have only partial insights. According to a hypothesis first formulated by Zimmermann (1983), water stress-induced cavitation is thought to occur when a tiny air bubble penetrates through a pit membrane, and would consequently be strongly influenced by the porosity of the membrane (Tyree and Sperry, 1988; Cochard, 2006). There is also experimental evidence for a role of the mechanical properties of the pit membrane in this cavitation process (Choat et al., 2004; Sperry and Hacke, 2004). Clearly, the structural, physical, and chemical properties of pit membranes are central to the determinism of cavitation.Pit membranes are modified primary cell walls made of tightly interwoven cellulose microfibrils in a matrix of hydrated hemicelluloses and pectins. Pectins consist of a complex set of GalUA (GalA)-rich polysaccharides, and four pectic domains can be distinguished: homogalacturonan (HG), rhamnogalacturonan I, rhamnogalacturonan II, and xylogalacturonan (Willats et al., 2001). The high degree of structural complexity and heterogeneity across the pectin family is the result of both biosynthesis in the endomembrane system and the action of an array of wall-based pectin-modifying enzymes (Willats et al., 2001). HG units are synthesized in the Golgi apparatus and deposited in the cell wall in a form containing 70% to 80% methyl-esterified GalA residues (O''Neill et al., 1990; Mohnen, 1999). The removal of methyl ester groups by pectin methyl esterase (PME) within the cell wall matrix produces free carboxyl groups capable of being cross-linked by calcium cations in an “egg-box” structure (Grant et al., 1973; Pelloux et al., 2007). These calcium-dependent cross-linkages are dependent both on the degree and the distribution of methyl-esterified GalA units through the HG network (Willats et al., 2001). Calcium therefore plays a central role as it determines the supramolecular assembly of the pectic chains and the formation of a pectate gel.Pectins capable of Ca²⁺ cross-linking are particularly common in bordered pit membranes (Chaffey et al., 1997; Hafren et al., 2000). Moreover, pectin-bound calcium influences wall elasticity (Ezaki et al., 2005; Proseus and Boyer, 2006; Derbyshire et al., 2007), and could therefore influence the stretching properties of the pit membranes and, consequently, the mechanism of cavitation. We tested the hypothesis that calcium plays a major role in the determinism of cavitation. This hypothesis was formulated long ago (Sperry and Tyree, 1988) but has not yet been thoroughly tested. We designed a series of experiments to demonstrate the specific role of calcium in this mechanism, and analyzed a large number of woody species to establish the role of calcium cross-linkage in across- and within-species variation in cavitation resistance. The data strongly support our hypothesis.     

Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila

Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP₃ 3-K, IP₃K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP₃Ks (IP₃K-1 and IP₃K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP₃K-1 directly increases H₂O₂ production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells–assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters–is significantly increased by IP₃K-1 under oxidative stress conditions, leading to apoptosis.The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP₃K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.     

Severe imported falciparum malaria: a cohort study in 400 critically ill adults

Background

Large studies on severe imported malaria in non-endemic industrialized countries are lacking. We sought to describe the clinical spectrum of severe imported malaria in French adults and to identify risk factors for mortality at admission to the intensive care unit.

Methodology and Principal Findings

Retrospective review of severe Plasmodium falciparum malaria episodes according to the 2000 World Health Organization definition and requiring admission to the intensive care unit. Data were collected from medical charts using standardised case-report forms, in 45 French intensive care units in 2000–2006. Risk factors for in-hospital mortality were identified by univariate and multivariate analyses.Data from 400 adults admitted to the intensive care unit were analysed, representing the largest series of severe imported malaria to date. Median age was 45 years; 60% of patients were white, 96% acquired the disease in sub-Saharan Africa, and 65% had not taken antimalarial chemoprophylaxis. Curative quinine treatment was used in 97% of patients. Intensive care unit mortality was 10.5% (42 deaths). By multivariate analysis, three variables at intensive care unit admission were independently associated with hospital death: older age (per 10-year increment, odds ratio [OR], 1.72; 95% confidence interval [95%CI], 1.28–2.32; P = 0.0004), Glasgow Coma Scale score (per 1-point decrease, OR, 1.32; 95%CI, 1.20–1.45; P<0.0001), and higher parasitemia (per 5% increment, OR, 1.41; 95%CI, 1.22–1.62; P<0.0001).

Conclusions and Significance

In a large population of adults treated in a non-endemic industrialized country, severe malaria still carried a high mortality rate. Our data, including predictors of death, can probably be generalized to other non-endemic countries where high-quality healthcare is available.     

Development of Sink Capacity of the "Storage Root" in a Radish Variety with a Low Ratio of "Storage Root" to Shoot

Controling mechanisms of sink capacity are poorly understood.Previously we suggested that sucrose synthase (SuSy), but notinvertase, plays an important role for sink capacity of theradish "storage root" in a variety, Raphanus sativus L. (cv.White Cherish) [plant Cell Physiol. (1999) 40: 369]. With thisvariety about 50% of the total dry weight (DW) was in the "storageroot" at 21 d after sowing (DAS). We investigated the sink capacityof another radish variety, R. sativus L. (cv. Kosena) with alow ratio of "storage root" to shoot. With the latter varietyonly 3% of the total DW was in the "storage root" at 21 DAS.Sink activity (increase in DW of the "storage root" per unitof DW present per unit of time) of the "storage root" in Kosenaas well as White Cherish was strongly related to the level andactivity of SuSy but not to the activity of invertase. Theseresults confirmed that SuSy rather than invertase may be criticalfor the development of the sink activity of the radish "storageroot" and that the reaction products of UDP-glucose and fructoseare utilized for sink growth including biosynthesis of the cellwall. In Kosena photosynthates seemed to be partitioned mainlyinto developing leaves and fibrous roots. Differences in partitioningof photosynthates among various sinks with these two varietiesare discussed including anatomical considerations. (Received July 19, 1999; Accepted September 30, 1999)