Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.     

The Synechocystis PCC6803 MerA-Like Enzyme Operates in the Reduction of Both Mercury and Uranium under the Control of the Glutaredoxin 1 Enzyme

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX₂C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.     

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Is local selection so widespread in river organisms? Fractal geometry of river networks leads to high bias in outlier detection

Identifying local adaptation is crucial in conservation biology to define ecotypes and establish management guidelines. Local adaptation is often inferred from the detection of loci showing a high differentiation between populations, the so‐called F_ST outliers. Methods of detection of loci under selection are reputed to be robust in most spatial population models. However, using simulations we showed that F_ST outlier tests provided a high rate of false‐positives (up to 60%) in fractal environments such as river networks. Surprisingly, the number of sampled demes was correlated with parameters of population genetic structure, such as the variance of F_STs, and hence strongly influenced the rate of outliers. This unappreciated property of river networks therefore needs to be accounted for in genetic studies on adaptation and conservation of river organisms.     

Wearable-Sensor-Based Classification Models of Faller Status in Older Adults

Wearable sensors have potential for quantitative, gait-based, point-of-care fall risk assessment that can be easily and quickly implemented in clinical-care and older-adult living environments. This investigation generated models for wearable-sensor based fall-risk classification in older adults and identified the optimal sensor type, location, combination, and modelling method; for walking with and without a cognitive load task. A convenience sample of 100 older individuals (75.5 ± 6.7 years; 76 non-fallers, 24 fallers based on 6 month retrospective fall occurrence) walked 7.62 m under single-task and dual-task conditions while wearing pressure-sensing insoles and tri-axial accelerometers at the head, pelvis, and left and right shanks. Participants also completed the Activities-specific Balance Confidence scale, Community Health Activities Model Program for Seniors questionnaire, six minute walk test, and ranked their fear of falling. Fall risk classification models were assessed for all sensor combinations and three model types: multi-layer perceptron neural network, naïve Bayesian, and support vector machine. The best performing model was a multi-layer perceptron neural network with input parameters from pressure-sensing insoles and head, pelvis, and left shank accelerometers (accuracy = 84%, F1 score = 0.600, MCC score = 0.521). Head sensor-based models had the best performance of the single-sensor models for single-task gait assessment. Single-task gait assessment models outperformed models based on dual-task walking or clinical assessment data. Support vector machines and neural networks were the best modelling technique for fall risk classification. Fall risk classification models developed for point-of-care environments should be developed using support vector machines and neural networks, with a multi-sensor single-task gait assessment.     

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.     

Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay

In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs). This is based on the elongation of 32P 5'-end-labeled oligonucleotide primers annealed to complementary oligonucleotide templates. Incorporation within the primer/template (p/t) was catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I under conditions where the concentration of the dNTP to be analyzed is limiting. Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together. Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane. This method allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI.     

Does clinical research provide any "direct individual benefit"?

The European Commission and Parliament have promulgated a directive on clinical research (2001/20/CE) in April 2001. Its provisions have to be incorporated in all national laws by May 2004. Accordingly, the French " loi Huriet " (a law passed in 1988 organizing clinical research in France) had to be revised. During the process, it appeared that a key issue was the suppression of a distinction made by this law between research with and without " direct individual benefit ", a French specificity, as the directive recommends rather the assessment of the risk/benefit ratio. In order to harmonize the French legislation with the other European laws, and to suppress a set of provisions which have been repeatedly attacked during the last ten years, the French members of parliament have voted on October 2003 a new law which, among other important modifications, suppress that distinction.     

Depletion of deoxyribonucleoside triphosphate pools in tumor cells by nitric oxide

Nitric oxide displays pro- and anti-tumor activities, prompting further studies to better understand its precise role. Nitric oxide inhibits ribonucleotide reductase (RnR), the limiting enzyme for de novo dNTP synthesis. We report here the first detailed analysis of dNTP variations induced in tumor cells by NO. NO prodrugs induced a depletion in dNTP pools and an activation of the pyrimidine salvage pathway, as did hydroxyurea, the prototypic RnR inhibitor. In the presence of dipyridamole, which blocked salvaged dNTP synthesis, depletion of dNTP pools was also observed in tumor cells cocultured with macrophages expressing the high-output iNOS activity. This effect was rapid, reversible, blocked by NO scavengers, and cGMP independent. It was quantitatively correlated to iNOS activity. In the absence of dipyridamole, NO still induced a decrease in dATP concentration in tumor cells cocultured with macrophages, whereas surprisingly, concentrations of dCTP and dTTP expanded considerably, resulting in a strong imbalance in dNTP pools. NO prodrugs did not cause such an increase in pyrimidine dNTP, suggesting that pyrimidine nucleosides were released by NO-injured macrophages. Altered dNTP levels have been reported to promote mutagenesis and apoptosis. It is suggested that abnormal changes in dNTP pools in tumors might contribute to NO-dependent toxicity.