Biodiversity in sulfur metabolism in hemiascomycetous yeasts

The evolution of the metabolism of sulfur compounds among yeast species was investigated. Differences between species were observed in the cysteine biosynthesis pathway. Most yeast species possess two pathways leading to cysteine production, the transsulfuration pathway and the O-acetyl-serine (OAS) pathway, with the exception of Saccharomyces cerevisiae and Candida glabrata, which only display the transsulfuration pathway, and Schizosaccharomyces pombe, which only have the OAS pathway. An examination of the components of the regulatory network in the different species shows that it is conserved in all the species analyzed, as its central component Met4p was shown to keep its functional domains and its partners were present. The analysis of the presence of genes involved in the catabolic pathway shows that it is evolutionarily conserved in the sulfur metabolism and leads us to propose a role for two gene families which appeared to be highly conserved. This survey has provided ways to understand the diversity of sulfur metabolism products among yeast species through the reconstruction of these pathways. This diversity could account for the difference in metabolic potentialities of the species with a biotechnological interest.     

The curious case of NG2 cells: transient trend or game changer?

It has been 10 years since the seminal work of Dwight Bergles and collaborators demonstrated that NG2 (nerve/glial antigen 2)-expressing oligodendrocyte progenitor cells (NG2 cells) receive functional glutamatergic synapses from neurons (Bergles et al., 2000), contradicting the old dogma that only neurons possess the complex and specialized molecular machinery necessary to receive synapses. While this surprising discovery may have been initially shunned as a novelty item of undefined functional significance, the study of neuron-to-NG2 cell neurotransmission has since become a very active and exciting field of research. Many laboratories have now confirmed and extended the initial discovery, showing for example that NG2 cells can also receive inhibitory GABAergic synapses (Lin and Bergles, 2004) or that neuron-to-NG2 cell synaptic transmission is a rather ubiquitous phenomenon that has been observed in all brain areas explored so far, including white matter tracts (Kukley et al., 2007; Ziskin et al., 2007; Etxeberria et al., 2010). Thus, while still being in its infancy, this field of research has already brought many surprising and interesting discoveries, and has become part of a continuously growing effort in neuroscience to re-evaluate the long underestimated role of glial cells in brain function (Barres, 2008). However, this area of research is now reaching an important milestone and its long-term significance will be defined by its ability to uncover the still elusive function of NG2 cells and their synapses in the brain, rather than by its sensational but transient successes at upsetting the old order established by neuronal physiology. To participate in the effort to facilitate such a transition, here we propose a critical review of the latest findings in the field of NG2 cell physiology – discussing how they inform us on the possible function(s) of NG2 cells in the brain – and we present some personal views on new directions the field could benefit from in order to achieve lasting significance.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2011-31 July 2011

This article documents the addition of 112 microsatellite marker loci and 24 pairs of single nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Agelaius phoeniceus, Austrolittorina cincta, Circus cyaneus, Circus macrourus, Circus pygargus, Cryptocoryne × purpurea Ridl. nothovar. purpurea, Mya arenaria, Patagioenas squamosa, Prochilodus mariae, Scylla serrata and Scytalopus speluncae. These loci were cross-tested on the following species: Cryptocoryne × purpurea nothovar. purpurea, Cryptocoryne affinis, Cryptocoryne ciliata, Cryptocoryne cordata var. cordata, Cryptocoryne elliptica, Cryptocoryne griffithii, Cryptocoryne minima, Cryptocoryne nurii and Cryptocoryne schulzei. This article also documents the addition of 24 sequencing primer pairs and 24 allele-specific primers or probes for Aphis glycines.     

Factors associated with negative direct sputum examination in Asian and African HIV-infected patients with tuberculosis (ANRS 1260)

Objective

To identify factors associated with negative direct sputum examination among African and Cambodian patients co-infected by Mycobacterium tuberculosis and HIV.

Design

Prospective multicenter study (ANRS1260) conducted in Cambodia, Senegal and Central African Republic.

Methods

Univariate and multivariate analyses (logistic regression) were used to identify clinical and radiological features associated with negative direct sputum examination in HIV-infected patients with positive M. tuberculosis culture on Lowenstein-Jensen medium.

Results

Between September 2002 and December 2005, 175 co-infected patients were hospitalized with at least one respiratory symptom and pulmonary radiographic anomaly. Acid-fast bacillus (AFB) examination was positive in sputum samples from 110 subjects (63%) and negative in 65 patients (37%). Most patients were at an advanced stage of HIV disease (92% at stage III or IV of the WHO classification) with a median CD4 cell count of 36/mm³. In this context, we found that sputum AFB negativity was more frequent in co-infected subjects with associated respiratory tract infections (OR = 2.8 [95%CI:1.1–7.0]), dyspnea (OR = 2.5 [95%CI:1.1–5.6]), and localized interstitial opacities (OR = 3.1 [95%CI:1.3–7.6]), but was less frequent with CD4≤50/mm³ (OR = 0.4 [95%CI:0.2–0.90), adenopathies (OR = 0.4 [95%CI:0.2–0.93]) and cavitation (OR = 0.1 [95%CI:0.03–0.6]).

Conclusions

One novel finding of this study is the association between concomitant respiratory tract infection and negative sputum AFB, particularly in Cambodia. This finding suggests that repeating AFB testing in AFB-negative patients should be conducted when broad spectrum antibiotic treatment does not lead to complete recovery from respiratory symptoms. In HIV-infected patients with a CD4 cell count below 50/mm3 without an identified cause of pneumonia, systematic AFB direct sputum examination is justified because of atypical clinical features (without cavitation) and high pulmonary mycobacterial burden.     

Positively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 β-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the β-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K₁/K₂ ≥ 5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K₂ < 80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its α-helical content, presumably associated with enhanced flexibility.     

Discovery and Characterization of an Arabidopsis thaliana N-Acylphosphatidylethanolamine Synthase

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D β/γ isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. β-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.N-Acylethanolamines (NAEs)² are bioactive lipids composed of an ethanolamine headgroup amide-linked to an acyl chain varying in length and degree of saturation. In animals, NAEs are involved in different physiological processes, such as neuroprotective action (1), embryo development (2), cell proliferation (3), apoptosis (4), nociception, anxiety, inflammation, appetite/anorexia, learning, and memory (for review, see Ref. 5). Most studies carried out with animal cells/tissues have focused on N-arachidonoylethanolamine (anandamide, NAE20:4), which is synthesized in brain neurons but also, under certain conditions, in macrophage cells (6). NAE20:4 binds CB1 cannabinoid receptors located in brain neurons (7) and also acts as ligand of vanilloid receptors for pain modulation (8). In addition, it has been shown that NAE20:4 also promotes food intake, whereas NAE18:0 and NAE18:1 exert anorexic effects by increasing satiety (9–11). NAE16:0 is accumulated during inflammation and has several anti-inflammatory effects (for a review, see Ref. 12).In plants, NAEs are thought to be involved in various physiological functions. For example, because NAE levels observed in various dry seeds decline rapidly after imbibition, a possible role of these compounds in the regulation of seed germination has been proposed (13). It was further observed that the addition of 25 μm NAE12:0 to growth medium of Arabidopsis thaliana leads to a decrease in the size of the main and lateral roots and in root hair formation. This reduction in growth was associated with a modification of cytoskeletal organization (14). NAE12:0 is also able to delay cut Dianthus caryophyllus (carnation) senescence by decreasing oxidative damage and enhancing antioxidant defense (15), whereas NAE14:0 inhibits the elicitor-induced medium alkalinization and activates phenylalanine ammonia lyase gene expression involved in plant defense against pathogen attack (16).Both in plant and animal cells (for a review, see Ref. 17), NAEs are formed by the hydrolysis (by PLDs) of N-acylphosphatidylethanolamine (NAPE). NAPE is an unusual derivative of phosphatidylethanolamine (PE) with a third fatty acid linked to the amine position of the ethanolamine headgroup. In animals, the formation of NAEs is catalyzed by a PLD with a high specificity toward NAPE (NAPE-PLD). In plants, PLDβ and PLDγ isoforms, but not PLDα, hydrolyzed NAPE into NAE in vitro, and this is thought to operate in response to several biotic and abiotic stresses. Both in animals and in plants, NAEs signaling is terminated by the action of fatty acid amide hydrolases, which hydrolyze NAEs to free fatty acid and ethanolamine. FAAH has been identified and characterized in mammals and plants (for a review, see Ref. 17). In Arabidopsis, FAAH has been shown to modulate NAE content. Moreover, lines overexpressing FAAH displayed enhanced seedling growth as well as increased cell size (18) and were also more susceptible to bacterial pathogens (19).Although the role of NAEs and their catabolism have been extensively investigated, little is known about their precursors, the NAPEs. NAPEs represent a minor phospholipid class but are present in all tissues of plants and animals. The principal function of NAPEs is to serve as a precursor for the production of lipid mediator NAEs, but it has also been suggested that NAPEs could serve as a membrane stabilizer to maintain cellular compartmentalization during tissue damage (20). More recently, N-palmitoyl-PE was proposed to act as an inhibitor of macrophage phagocytosis through inhibition of the activation of Rac1 and Cdc42 (21).In the animal and plant kingdoms, therefore, the signaling events mediated by NAEs appear to be involved in many physiological processes that have been extensively studied. The genes encoding the enzymes involved in the synthesis (from NAPEs) and the degradation of NAEs have been cloned and characterized. By contrast, little is known about the physiological roles of NAPEs or about the first step of this lipid signaling pathway, namely the N-acylation of PE to form NAPEs. In animals, the synthesis of NAPEs is catalyzed by an N-acyltransferase, where the O-linked acyl unit from a phospholipid donor is transferred to the ethanolamine headgroup of PE (22). Recently, a rat LRAT-like protein 1 or RLP1 was shown to display such an activity, but according to the authors, RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca²⁺-dependent N-acyltransferase (23). However, a different situation is observed in plants. NAPE synthase activity was shown to directly acylate PE with free fatty acids (24, 25), but a gene encoding a NAPE synthase activity remained unidentified until now. The present work shows that the A. thaliana acyltransferase At1g78690p catalyzes the synthesis of NAPEs from PE and acyl-CoAs in vitro as well as in vivo when this enzyme is expressed in E. coli and overexpressed in plants.     

Development of methods for quantitative comparison of pooled shRNAs by mass sequencing

Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments.     

Stabilized hemiacetal complexes as precursors for the controlled release of bioactive volatile alcohols

Hemiacetals of pyridine-2-carbaldehyde derivatives and volatile alcohols can be stabilized in organic solution in the presence of protons or different metal cations. Despite the inherent instability of hemiacetals in H(2) O, stabilizing them with zinc(II) triflate and adding them to a cationic surfactant formulation resulted in the slow release of the alcohol from cotton surfaces being treated with the hemiacetal complex. Stabilized hemiacetals might thus be suitable delivery systems of bioactive volatiles by rapid hydrolysis in H(2) O-based media.