Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.     

Jupiter, a new Drosophila protein associated with microtubules

In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.     

Combined effect of prenatal solvent exposure and GSTT1 or GSTM1 polymorphisms in the risk of birth defects

Exposure to solvents during pregnancy has long been suspected to increase the risk of congenital malformations. Glutathione S-transferases (GSTs) are enzymes essential for the detoxification of various chemicals. Our objective here was to assess whether GST polymorphisms might modify the association between maternal solvent exposure and the risk of birth defects. A prospective cohort included 3421 pregnant women in Brittany, France (2002-2006). Occupational exposure to solvents was assessed from a job-exposure matrix. Congenital malformations were diagnosed among livebirths, stillbirths, and medical pregnancy terminations. Using a nested case-control design, 32 babies with major birth defects were compared to 348 normal births for babies' cord blood genotypes (at GSTT1 and GSTM1) and maternal occupational solvent exposure. Logistic models were used to adjust for potential confounders. The risk of major defects increased significantly in women with solvent exposure (20% of controls and 34% of cases). Frequencies of the null genotype of both the GSTT1 and GSTM1 genes were similar among controls and cases. There was a significantly increased risk of birth defects in GSTM1 not-null cord-blood genotype in pregnancies exposed to solvents (odds ratio [OR], 1.0 for not-null, not-exposed; OR, 4.0 for not-null, exposed; 95% confidence interval [CI], 1.4-11.2; OR, 1.6 for null, not-exposed; 95% CI, 0.6-3.9; OR, 1.0 for null, exposed; 95% CI, 0.2-4.7; p = 0.05). This nested case-control study suggests that the child's GSTM1 genotype modifies the risk of major birth defects among offspring of solvent-exposed women. Replication and additional investigations are necessary to confirm and elucidate these findings.     

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

ABSTRACT: BACKGROUND: Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. RESULTS: The genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW) of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under employed conditions. CONCLUSIONS: Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.     

Influence of marital status and employment status on long-term adherence with continuous positive airway pressure in sleep apnea patients

Background

Long-term adherence is a major issue in patients receiving home continuous positive airway pressure (CPAP) therapy for obstructive sleep apnea-hypopnea syndrome (OSAHS). In a multicenter prospective cohort (the Institut de Recherche en Santé Respiratoire des Pays de la Loire [IRSR] sleep cohort) of consecutive OSAHS patients in whom CPAP had been prescribed for at least 90 days, we studied the impact on long-term treatment adherence of socioeconomic factors, patients and disease characteristics prior to CPAP initiation.

Methods and Principal Findings

Among 1,141 patients in whom CPAP had been prescribed for an average of 504±251 days (range: 91 to 1035), 674 (59%) were adherent with a mean daily use of CPAP≥4 h (mean: 6.42±1.35 h). Stepwise regression analysis identified 4 independent factors of CPAP adherence including apnea-hypopnea index (AHI) (OR: 1.549, 95%CI 1.163 to 2.062 for AHI≥30 vs. AHI<30; p = 0.003), body mass index (BMI) (OR: 1.786, 95%CI 1.131 to 2.822 for BMI≥25 and <30 kg/m², p = 0.01; OR: 1.768, 95%CI 1.145–2.731 for BMI≥30 kg/m², p = 0.01 vs. BMI<25 kg/m²), employment status (OR: 1.414, 95%CI 1.097–1.821 for retired vs. employed; p = 0.007) and marital status (OR: 1.482, 95%CI 1.088–2.019 for married or living as a couple vs. living alone; p = 0.01). Age, gender, Epworth sleepiness scale, depressive syndrome, associated cardiovascular morbidities, educational attainment and occupation category did not influence CPAP adherence.

Conclusions

Marital status and employment status are independent factors of CPAP adherence in addition to BMI and disease severity. Patients living alone and/or working patients are at greater risk of non-adherence, whereas adherence is higher in married and retired patients. These findings suggest that the social context of daily life should be taken into account in risk screening for CPAP non-adherence. Future interventional studies targeting at-risk patients should be designed to address social motivating factors and work-related barriers to CPAP adherence.     

Discovery of novel methanone derivatives acting as antimycobacterial agents

A series of pyrazoline derivatives were synthesized and in vitro activity against Mycobacterium tuberculosis H37Rv was carried out. Among the synthesized compounds, compounds (4d) and (4f) 4-aminophenyl-3-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2,3,3a,4-tetrahydroindeno[1,2-c]pyrazol-2-ylmethanone and 4-aminophenyl-6,7-dimethoxy-3-phenyl-2,3,3a,4-tetrahydroindeno[1,2-c]pyrazol-2-ylmethanone were found to be the most active agent against M. tuberculosis H37Rv with a minimum inhibitory concentration of 10 μg/mL.     

DEVELOPMENT OF SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS FROM INTERSIMPLE SEQUENCE REPEAT FINGERPRINTS FOR THE MOLECULAR DETECTION OF TOXIC PHYTOPLANKTON ALEXANDRIUM CATENELLA (DINOPHYCEAE) AND PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA (BACILLARIOPHYCEAE) FROM FRENCH COASTAL WATERS1

Harmful algal blooms are a serious threat to shellfish farming and human health all over the world. The monitoring of harmful algae in coastal waters originally involved morphological identification through microscopic examinations, which was often difficult unless performed by specialists and even then often did not permit identification of toxic species. More recently, specific molecular markers have been used to identify specific phytoplankton species or strains. Here we report on the use of the intersimple sequence repeat (ISSR) technique to develop specific sequence characterized amplified region markers (SCAR) and to identify with these tools two toxic species in French coastal waters, the diatom Pseudo‐nitzschia pseudodelicatissima (Hasle) Hasle and the dinoflagellate Alexandrium catenella (Whedon and Kofoid 1936), Balech 1985. Six polymorphic ISSR regions were selected among amplified fingerprints of a representative sample of phytoplankton species. After cloning and sequencing the selected polymorphic ISSR regions, pairs of internal primers were designed to amplify a unique and specific sequence designed as a SCAR marker. Of the six selected SCAR markers, three were specific to P. pseudodelicatissima and one for A. catenella. The SCAR marker specificity was confirmed by using basic local alignment search tool comparison, by experimental assays on different strains from 11 countries, and by checking that the sequence amplified was the expected one. When tested on water samples collected along the French shores, the four specific SCAR markers proved to be efficient tools for fast and low‐cost detection of toxic phytoplankton species.