Crystallographic studies on two bioisosteric analogues, N-acetyl-beta-D-glucopyranosylamine and N-trifluoroacetyl-beta-D-glucopyranosylamine, potent inhibitors of muscle glycogen phosphorylase

Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of beta-D-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-beta-D-glucopyranosylamine (NAG) (K(i) = 32 microM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 angstroms resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-beta-D-glucopyranosylamine (NFAG), with a K(i) = 75 microM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 angstroms resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 angstroms). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the K(i) values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.     

Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine

Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the alpha-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the alpha-subunit of GDH, increase in immunoreactive alpha-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive alpha-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.     

Heat acclimatization and hydration status of American football players during initial summer workouts

This investigation evaluated the new National Collegiate Athletic Association model of heat acclimatization for football players using physiological, psychological, fluid balance, anthropometric, and nutritional variables. Eleven football players (20 +/- 1 year, 1.88 +/- 0.05 m, and 115.36 +/- 18.85 kg) from a Division I football team were observed for the first 8 days of preseason practices. Measurements such as heart rate and gastrointestinal temperature (T(GI)) via telemetric sensor were taken before, 3 times during, and after practice daily. An average 1.39-kg (1.2%) decrease of body mass occurred from prepractice to postpractice (p < 0.01). Consistent with mild body mass losses, urinary indices of hydration status (i.e., color, specific gravity, and osmolality) indicated mild fluid deficits. A significant increase (p < 0.05) from pre- to postpractice was observed in urine color and urine specific gravity, but chronic hypohydration over the 8 days was not noted. The Environmental Symptoms Questionnaire (ESQ) postpractice score was significantly higher (p < 0.05) than the prepractice score was, but averages did not differ across practice days. There was no difference in postpractice T(GI) measurements across days (p < 0.05). Heart rate, T(GI), and ESQ measurements indicated that football players experienced gradual heat acclimatization and enhanced heat tolerance, despite progressive increases of exercise variables, clothing, and environmental stressors.     

Efficacy and acceptability of selective serotonin reuptake inhibitors for the treatment of depression in Parkinson's disease: a systematic review and meta-analysis of randomized controlled trials

Background  

Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed antidepressants for the treatment of depression in patients with Parkinson's Disease (PD) but data on their efficacy are controversial.     

Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers

Protist diversity is currently a much debated issue in eukaryotic microbiology. Recent evidence suggests that morphological and genetic diversity might be decoupled in some groups of protists, including ciliates, and that these organisms might be much more diverse than their morphology implies. We sought to assess the genetic and morphological diversity of Carchesium polypinum, a widely distributed peritrich ciliate. The mitochondrial marker cytochrome c oxidase subunit I and the nuclear small subunit ribosomal RNA were used to examine genetic diversity. For the morphological assessment, live microscopy and Protargol staining were used. The mitochondrial marker revealed six robust, deeply diverging, and strongly supported clades, while the nuclear gene was congruent for three of these clades. There were no major differences among individuals from the different clades in any of the morphological features examined. Thus, the underlying genetic diversity in C. polypinum is greater than what its morphology suggests, indicating that morphology and genetics are not congruent in this organism. Furthermore, because the clades identified by the mitochondrial marker are so genetically diverse and are confirmed by a conserved nuclear marker in at least three cases, we propose that C. polypinum be designated as a "cryptic species complex." Our results provide another example where species diversity can be underestimated in microbial eukaryotes when using only morphological criteria to estimate species richness.     

Stimulus sampling as an exploration mechanism for fast reinforcement learning

Reinforcement learning in neural networks requires a mechanism for exploring new network states in response to a single, nonspecific reward signal. Existing models have introduced synaptic or neuronal noise to drive this exploration. However, those types of noise tend to almost average out—precluding or significantly hindering learning —when coding in neuronal populations or by mean firing rates is considered. Furthermore, careful tuning is required to find the elusive balance between the often conflicting demands of speed and reliability of learning. Here we show that there is in fact no need to rely on intrinsic noise. Instead, ongoing synaptic plasticity triggered by the naturally occurring online sampling of a stimulus out of an entire stimulus set produces enough fluctuations in the synaptic efficacies for successful learning. By combining stimulus sampling with reward attenuation, we demonstrate that a simple Hebbian-like learning rule yields the performance that is very close to that of primates on visuomotor association tasks. In contrast, learning rules based on intrinsic noise (node and weight perturbation) are markedly slower. Furthermore, the performance advantage of our approach persists for more complex tasks and network architectures. We suggest that stimulus sampling and reward attenuation are two key components of a framework by which any single-cell supervised learning rule can be converted into a reinforcement learning rule for networks without requiring any intrinsic noise source. This work was supported by the Swiss National Science Foundation grant K-32K0-118084.     

Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2

In this study, we investigate the interplay between Ku, a central non‐homologous end‐joining component, and the Mre11–Rad50–Xrs2 (MRX) complex and Sae2, end‐processing factors crucial for initiating 5′‐3′ resection of double‐strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication‐associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end‐processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.     

Detection of metallo-β-lactamase genes in clinical specimens by a commercial multiplex PCR system

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-β-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting bla_VIM genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.