Comparative characterization of novel ene‐reductases from cyanobacteria

The growing importance of biocatalysis in the syntheses of enantiopure molecules results from the benefits of enzymes regarding selectivity and specificity of the reaction and ecological issues of the process. Ene‐reductases (ERs) from the old yellow enzyme family have received much attention in the last years. These flavo‐enzymes catalyze the trans‐specific reduction of activated C?C bonds, which is an important reaction in asymmetric synthesis, because up to two stereogenic centers can be created in one reaction. However, limitations of ERs described in the literature such as their moderate catalytic activity and their strong preference for NADPH promote the search for novel ERs with improved properties. In this study, we characterized nine novel ERs from cyanobacterial strains belonging to different taxonomic orders and habitats. ERs were identified with activities towards a broad spectrum of alkenes. The reduction of maleimide was catalyzed with activities of up to 35.5 U mg^?1 using NADPH. Ketoisophorone and (R)‐carvone, which were converted to the highly valuable compounds (R)‐levodione and (2R,5R)‐dihydrocarvone, were reduced with reaction rates of up to 2.2 U mg^?1 with NADPH. In contrast to other homologous ERs from the literature, NADH was accepted at moderate to high rates as well: Enzyme activities of up to 16.7 U mg^?1 were obtained for maleimide and up to 1.3 U mg^?1 for ketoisophorone and (R)‐carvone. Additionally, excellent stereoselectivities were achieved in the reduction of (R)‐carvone (97–99% de). In particular, AnabaenaER3 from Anabaena variabilis ATCC 29413 and AcaryoER1 from Acaryochloris marina MBIC 11017 were identified as useful biocatalysts. Therefore, novel ERs from cyanobacteria with high catalytic efficiency were added to the toolbox for the asymmetric reduction of alkenes. Biotechnol. Bioeng. 2013; 110: 1293–1301. © 2012 Wiley Periodicals, Inc.     

The Pluripotency Factor-Bound Intron 1 of Xist Is Dispensable for X Chromosome Inactivation and Reactivation In Vitro and In Vivo

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Association of a common TLR-6 polymorphism with coronary artery disease – implications for healthy ageing?

Background

The pro-inflammatory status of the elderly triggers most of the age-related diseases such as cancer and atherosclerosis. Atherosclerosis, the leading cause world wide of morbidity and death, is an inflammatory disease influenced by life-style and genetic host factors. Stimuli such as oxLDL or microbial ligands have been proposed to trigger inflammation leading to atherosclerosis. It has recently been shown that oxLDL activates immune cells via the Toll-like receptor (TLR) 4/6 complex. Several common single nucleotide polymorphisms (SNPs) of the TLR system have been associated with atherosclerosis. To investigate the role of TLR-6 we analyzed the association of the TLR-6 SNP Pro249Ser with atherogenesis.

Results

Genotyping of two independent groups with CAD, as well as of healthy controls revealed a significant association of the homozygous genotype with a reduced risk for atherosclerosis (odds ratio: 0.69, 95% CI 0.51-0.95, P?=?0.02). In addition, we found a trend towards an association with the risk of restenosis after transluminal coronary angioplasty (odds ratio: 0.53, 95% CI 0.24-1.16, P?=?0.12). In addition, first evidence is presented that the frequency of this protective genotype increases in a healthy population with age. Taken together, our results define a role for TLR-6 and its genetic variations in modulating the inflammatory response leading to atherosclerosis.

Conclusions

These results may lead to a better risk stratification, and potentially to an improved prophylactic treatment of high-risk populations. Furthermore, the protective effect of this polymorphism may lead to an increase of this genotype in the healthy elderly and may therefore be a novel genetic marker for the well-being during aging.

     

Testing Species Delimitations in Four Italian Sympatric Leuciscine Fishes in the Tiber River: A Combined Morphological and Molecular Approach

Leuciscine fishes represent an important component of freshwater ichthyofauna endemic to northern Mediterranean areas. This lineage shows high intra-specific morphological variability and exhibits high levels of hybridization, two characteristics that contribute to systematic uncertainties, misclassification of taxa and, potentially, the mismanagement of biodiversity. This study focused on brook chub, Squalius lucumonis, an endemic taxon of Central Italy. The taxonomic status of this species has long been questioned, and a hybrid origin from sympatric leusciscines (S. squalus x Rutilus rubilio, or S. squalus x Telestes muticellus) has been hypothesised. A phenotypic (evaluating shape and meristic counts) and genetic (using mitochondrial and nuclear markers) investigation of these four taxa was conducted to test species delimitation in sympatric areas and to evaluate the taxonomic status of S. lucumonis. One hundred and forty-five individuals of all four taxa were collected within streams of the lowest portion of the Tiber River basin and analysed; this region encompasses a large portion of the S. lucumonis distribution. The different morphological and genetic approaches were individually examined, compared, and then combined in a quantitative model to both investigate the limits of each approach and to identify cases of misclassification. The results obtained confirm the cladogenetic non-hybrid origin of S. lucumonis, highlight the need for immediate conservation actions and emphasise the value of an integrated approach in the study of leuciscines evolution.     

Dose-Dependent Induction of Murine Th1/Th2 Responses to Sheep Red Blood Cells Occurs in Two Steps: Antigen Presentation during Second Encounter Is Decisive

The differentiation of CD4 T cells into Th1 and Th2 cells in vivo is difficult to analyze since it is influenced by many factors such as genetic background of the mice, nature of antigen, and adjuvant. In this study, we used a well-established model, which allows inducing Th1 or Th2 cells simply by low (LD, 10⁵) or high dose (HD, 10⁹) injection of sheep red blood cells (SRBC) into C57BL/6 mice. Signature cytokine mRNA expression was determined in specific splenic compartments after isolation by laser-microdissection. LD immunization with SRBC induced T cell proliferation in the splenic T cell zone but no Th1 differentiation. A second administration of SRBC into the skin rapidly generated Th1 cells. In contrast, HD immunization with SRBC induced both T cell proliferation and immediate Th2 differentiation. In addition, splenic marginal zone and B cell zone were activated indicating B cells as antigen presenting cells. Interestingly, disruption of the splenic architecture, in particular of the marginal zone, abolished Th2 differentiation and led to the generation of Th1 cells, confirming that antigen presentation by B cells directs Th2 polarization. Only in its absence Th1 cells develop. Therefore, B cells might be promising targets in order to therapeutically modulate the T cell response.     

Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.     

Alleged Approach-Avoidance Conflict for Food Stimuli in Binge Eating Disorder

ObjectiveFood stimuli are omnipresent and naturally primary reinforcing stimuli. One explanation for the intake of high amounts of food in binge eating disorder (BED) is a deviant valuation process. Valuation of food stimuli is supposed to influence approach or avoidance behaviour towards food. Focusing on self-reported and indirect (facial electromyography) valuation process, motivational aspects in the processing of food stimuli were investigated.MethodsWe compared an overweight sample with BED (BED+) with an overweight sample without BED (BED-) and with normal weight controls (NWC) regarding their self-reported and indirect (via facial electromyography) valuation of food versus non-food stimuli.ResultsRegarding the self-reported valuation, the BED+ sample showed a significantly stronger food-bias compared to the BED- sample, as food stimuli were rated as significantly more positive than the non-food stimuli in the BED+ sample. This self-reported valuation pattern could not be displayed in the indirect valuation. Food stimuli evoked negative indirect valuation in all groups. The BED+ sample showed the plainest approach-avoidance conflict marked by a diverging self-reported (positive) and indirect (negative) valuation of food stimuli.ConclusionsBED+ showed a deviant self-reported valuation of food as compared to BED-. The valuation process of the BED+ sample seems to be characterized by a motivational ambivalence. This ambivalence should be subject of further studies and may be of potential use for therapeutic interventions.     

Factors that Influence the Formation and Stability of Thin,Cryo-EM Specimens

Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.     

A method for in situ monitoring of the isotope composition of tree xylem water using laser spectroscopy

Field studies analyzing the stable isotope composition of xylem water are providing important information on ecosystem water relations. However, the capacity of stable isotopes to characterize the functioning of plants in their environment has not been fully explored because of methodological constraints on the extent and resolution at which samples could be collected and analysed. Here, we introduce an in situ method offering the potential to continuously monitor the stable isotope composition of tree xylem water via its vapour phase using a commercial laser‐based isotope analyser and compact microporous probes installed into the xylem. Our technique enables efficient high‐frequency measurement with intervals of only a few minutes per sample while eliminating the need for costly and cumbersome destructive collection of plant material and laboratory‐based processing. We present field observations of xylem water hydrogen and oxygen isotope compositions obtained over several days including a labelled irrigation event and compare them against results from concurrent destructive sampling with cryogenic distillation and mass spectrometric analysis. The data demonstrate that temporal changes as well as spatial patterns of integration in xylem water isotope composition can be resolved through direct measurement. The new technique can therefore present a valuable tool to study the hydraulic architecture and water utilization of trees.     

Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-β2

To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor β 2 (TGF-β₂) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-β₂-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/?TGF-β₂ revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-β₂ and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-β₂ mRNA expression (qRT-PCR), TGF-β₂ protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-β₂-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.