In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

Structure of the Pentameric Ligand-Gated Ion Channel GLIC Bound with Anesthetic Ketamine

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Highlights? The structure of ketamine-bound GLIC reveals an anesthetic binding site ? The study provides compelling evidence for allosteric inhibition by anesthetics ? Ketamine inhibition on GLIC is similar to competitive antagonist action on nAChRs ? Ketamine directly acts on pLGICs in addition to NMDA receptors     

Substrate docking and molecular dynamic simulation for prediction of fungal enzymes from Trichoderma species-assisted extraction of nanocellulose from oil palm leaves

Abstract

Fungi of the Trichoderma species are valued industrial enzymes in support of the ‘zero-waste’ technology to convert agro-industrial biomass into valuable products, i.e. nanocellulose (NC). In this study, an in silico approach using substrate docking and molecular dynamic (MD) simulation was used to predict the order of which the multilayers of cellulosic polymers, i.e. lignin, hemicellulose and cellulose in oil palm leaves (OPL) are degraded by fungal enzymes, endocellulase and exocellulase. The study aimed to establish the catalytic tendencies of the enzymes to optimally degrade the cellulosic components of OPL for high yield production of NC. Energy minimized endocellulase and exocellulase models revealed satisfactory scores of PROCHECK (90.0% and 91.2%), Verify3D (97.23% and 98.85%) and ERRAT (95.24% and 91.00%) assessments. Active site prediction by blind docking, COACH meta-server and multiple sequence alignment indicated the catalytic triads for endocellulase and exocellulase were Ser116–His205–Glu249 and Ser382–Arg124–Asp385, respectively. Binding energy of endocellulase docked with hemicellulose (?6.0 ? kcal mol^?1) was the most favourable followed by lignin (?5.6 ? kcal mol^?1) and cellulose (?4.4 ? kcal mol^?1). Exocellulase, contrarily, bonded favorably with lignin (?8.7 ? kcal mol^?1), closely followed by cellulose (?8.5 ? kcal mol^?1) and hemicellulose (?8.4 ? kcal mol^?1). MDs simulations showed that interactions of complexes, endocellulase–hemicellulose and the exocellulase–cellulose being the most stable. Thus, the findings of the study successfully identified the specific actions of sugar-acting enzymes for NC production.

Communicated by Ramaswamy H. Sarma     

Gas7b (Growth Arrest Specific Protein 7b) Regulates Neuronal Cell Morphology by Enhancing Microtubule and Actin Filament Assembly

Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.     

Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris.

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1). Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1). This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.     

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

A Novel Role for hGas7b in Microtubular Maintenance: POSSIBLE IMPLICATION IN TAU-ASSOCIATED PATHOLOGY IN ALZHEIMER DISEASE*

Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.     

Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.     

On following behaviour as a mechanism for collective movement

During collective movement, animals display a wide variety of mechanisms to maintain cohesion. In some species, individuals rely mainly on following their direct predecessor, thereby forming spectacular processions of individuals in single file. Despite being the simplest case of following behaviour, it is largely absent from the theoretical literature on collective migrations. The objective of this study is to quantify the efficiency of following the predecessor, in terms of ensuring cohesion. The situation we consider is a sequence of individuals facing a bifurcation. The choice between left and right is influenced by the choice of the predecessor. First, we model this situation with a two-state Markov chain with a symmetric transition matrix. Cohesion is quantified as the expected number of individuals on either side, and the expected number of consecutive followers. Although cohesion increases with the probability of following, it remains surprisingly low unless the probability is almost equal to one. Furthermore, cohesion decreases with group size regardless of the probability of following. Then, we generalise our model to situations in which individuals have a preference for one of the two choices (asymmetric transition matrix). For some parameter sets, the tendency to follow each other leads a large fraction of the individuals to the non preferred side. Moreover, this fraction increases with the total population size. Finally, we include the possibility to follow N individuals. This provides the link between our model and other collective migration models. If enough individuals are perceived, the results shift from symmetrical (low cohesion) to asymmetrical (high cohesion) distribution of the individuals. All in all, our results suggest that following the direct predecessor must be complemented with other cohesive behaviours (involving the perception of more individuals or a navigation system) to guarantee its efficiency. We discuss our findings in the context of the different following behaviours covered in the literature.