Prebiotic processes in planetary atmospheres

Within 40 years of experimental studies in prebiotic chemistry, most of the building blocks of the living systems have been synthesized in plausible conditions of the primitive Earth. The starting ingredients correspond to two complementary classes: volatile organics, and their non volatile oligomers. They may have been formed in the atmosphere on the primitive Earth and/or imported by extra-terrestrial sources. Organic chemistry is involved in meteorites, comets, in the giant planets and several of their satellites. Again this chemistry presents the two complementary aspects. In particular, with a dense reduced atmosphere rich in organic compounds in gas and aerosol phases, Titan appears as a natural laboratory for studying prebiotic chemistry at a planetary scale.     

Adipose tissue proadipogenic redox changes in obesity

The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (alpha-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process.     

Identification and Differentiation of Legionella pneumophila and Legionella spp. with Real-Time PCR Targeting the 16S rRNA Gene and Species Identification by mip Sequencing

Fluorescent resonance energy transfer probes targeting the 16S rRNA gene were constructed for a sensitive and specific real-time PCR for identification and differentiation of Legionella pneumophila from other Legionella spp. For identification of non-L. pneumophila spp. by direct amplicon sequencing, two conventional PCR assays targeting the mip gene were established.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography

Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations. We developed a hydroxyapatite chromatography for the separation of both HDL-associated proteins. We confirmed that: (1) HPBP is strongly associated to PON1 in HDL, and generally both proteins are co-purified; (2) standard purification protocols of PON1 lead to impure enzyme; (3) hydroxyapatite chromatography allows the simultaneous purification of PON1 and HPBP.     

Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M^− 1 s^− 1, for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M^− 1 s^− 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M^− 1 s^− 1. In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.