Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.     

Computer aided screening and evaluation of herbal therapeutics against MRSA infections

Methicillin resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life threatening outbreaks such as community-onset and nosocomial infections has emerged as 'superbug'. The organism developed resistance to all classes of antibiotics including the best known Vancomycin (VRSA). Hence, there is a need to develop new therapeutic agents. This study mainly evaluates the potential use of botanicals against MRSA infections. Computer aided design is an initial platform to screen novel inhibitors and the data finds applications in drug development. The drug-likeness and efficiency of various herbal compounds were screened by ADMET and docking studies. The virulent factor of most of the MRSA associated infections are Penicillin Binding Protein 2A (PBP2A) and Panton-Valentine Leukocidin (PVL). Hence, native structures of these proteins (PDB: 1VQQ and 1T5R) were used as the drug targets. The docking studies revealed that the active component of Aloe vera, β-sitosterol (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- [(2R, 5R)-5-ethyl-6-methylheptan-2-yl] -10, 13-dimethyl 2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17- dodecahydro-1H-cyclopenta [a] phenanthren-3-ol) showed best binding energies of -7.40 kcal/mol and -6.34 kcal/mol for PBP2A and PVL toxin, respectively. Similarly, Meliantriol (1S-1-[ (2R, 3R, 5R)-5-hydroxy-3-[(3S, 5R, 9R, 10R, 13S, 14S, 17S)-3-hydroxy 4, 4, 10, 13, 14-pentamethyl-2, 3, 5, 6, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta[a] phenanthren-17-yl] oxolan-2-yl] -2- methylpropane-1, 2 diol), active compound in Azadirachta indica (Neem) showed the binding energies of -6.02 kcal/mol for PBP2A and -8.94 for PVL toxin. Similar studies were conducted with selected herbal compound based on pharmacokinetic properties. All in silico data tested in vitro concluded that herbal extracts of Aloe-vera, Neem, Guava (Psidium guajava), Pomegranate (Punica granatum) and tea (Camellia sinensis) can be used as therapeutics against MRSA infections.     

Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies

Formation of a noncanonical base pair between dFTP, a dTTP analogue that cannot form H bonds, and the fluorescent base analogue 2-aminopurine (2AP) was studied in order to discover how the bacteriophage T4 DNA polymerase selects nucleotides with high accuracy. Changes in 2AP fluorescence intensity provided a spectroscopic reporter of the nucleotide binding reactions, which were combined with rapid-quench, pre-steady-state reactions to measure product formation. These studies supported and extended previous findings that the T4 DNA polymerase binds nucleotides in multiple steps with increasing selectivity. With 2AP in the template position, initial dTTP binding was rapid but selective: K(d(dTTP)) (first step) = 31 microM; K(d(dCTP)) (first step) approximately 3 mM. In studies with dFTP, this step was revealed to have two components: formation of an initial preinsertion complex in which H bonds between bases in the newly forming base pair were not essential, which was followed by formation of a final preinsertion complex in which H bonds assisted. The second nucleotide binding step was characterized by increased discrimination against dTTP binding opposite template 2AP, K(d) (second step) = 367 microM, and additional conformational changes were detected in ternary enzyme-DNA-dTTP complexes, as expected for forming closed complexes. We demonstrate here that the second binding step occurs before formation of the phosphodiester bond. Thus, the high fidelity of nucleotide insertion by T4 DNA polymerase is accomplished by the sequential application of selectivity in first forming accurate preinsertion complexes, and then additional conformational changes are applied that further increase discrimination against incorrect nucleotides.     

The role of living/controlled radical polymerization in the formation of improved imprinted polymers

In this work, living/controlled radical polymerization (LRP) is compared with conventional free radical polymerization in the creation of highly and weakly cross-linked imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) networks. It elucidates, for the first time, the effect of LRP on the chain level and begins to explain why the efficiency of the imprinting process is improved using LRP. Imprinted polymers produced via LRP exhibited significantly higher template affinity and capacity compared with polymers prepared using conventional methods. The use of LRP in the creation of highly cross-linked imprinted polymers resulted in a fourfold increase in binding capacity without a decrease in affinity; whereas weakly cross-linked gels demonstrated a nearly threefold increase in binding capacity at equivalent affinity when LRP was used. In addition, by adjusting the double bond conversion, we can choose to increase either the capacity or the affinity in highly cross-linked imprinted polymers, thus allowing the creation of imprinted polymers with tailorable binding parameters. Using free radical polymerization in the creation of polymer chains, as the template-monomer ratio increased, the average molecular weight of the polymer chains decreased despite a slight increase in the double bond conversion. Thus, the polymer chains formed were shorter but greater in number. Using LRP neutralized the effect of the template. The addition of chain transfer agent resulted in slow, uniform, simultaneous chain growth, resulting in the formation of longer more monodisperse chains. Reaction analysis revealed that propagation time was extended threefold in the formation of highly cross-linked polymers when LRP techniques were used. This delayed the transition to the diffusion-controlled stage of the reaction, which in turn led to the observed enhanced binding properties, decreased polydispersity in the chains, and a more homogeneous macromolecular architecture.     

Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats

Wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. This end is achieved mainly by the synthesis of the connective tissue matrix. Collagen is the major protein of the extracellular matrix, and is the component which ultimately contributes to wound strength. In this work, we report the influence of Aloe vera on the collagen content and its characteristics in a healing wound. It was observed that Aloe vera increased the collagen content of the granulation tissue as well as its degree of crosslinking as seen by increased aldehyde content and decreased acid solubility. The type I/type III collagen ratio of treated groups were lower than that of the untreated controls, indicating enhanced levels of type III collagen. Wounds were treated either by topical application or oral administration of Aloe vera to rats and both treatments were found to result in similar effects.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

Phylogeny in Defining Model Plants for Lignocellulosic Ethanol Production: A Comparative Study of Brachypodium distachyon,Wheat, Maize,and Miscanthus x giganteus Leaf and Stem Biomass

The production of ethanol from pretreated plant biomass during fermentation is a strategy to mitigate climate change by substituting fossil fuels. However, biomass conversion is mainly limited by the recalcitrant nature of the plant cell wall. To overcome recalcitrance, the optimization of the plant cell wall for subsequent processing is a promising approach. Based on their phylogenetic proximity to existing and emerging energy crops, model plants have been proposed to study bioenergy-related cell wall biochemistry. One example is Brachypodium distachyon, which has been considered as a general model plant for cell wall analysis in grasses. To test whether relative phylogenetic proximity would be sufficient to qualify as a model plant not only for cell wall composition but also for the complete process leading to bioethanol production, we compared the processing of leaf and stem biomass from the C₃ grasses B. distachyon and Triticum aestivum (wheat) with the C₄ grasses Zea mays (maize) and Miscanthus x giganteus, a perennial energy crop. Lambda scanning with a confocal laser-scanning microscope allowed a rapid qualitative analysis of biomass saccharification. A maximum of 108–117 mg ethanol·g⁻¹ dry biomass was yielded from thermo-chemically and enzymatically pretreated stem biomass of the tested plant species. Principal component analysis revealed that a relatively strong correlation between similarities in lignocellulosic ethanol production and phylogenetic relation was only given for stem and leaf biomass of the two tested C₄ grasses. Our results suggest that suitability of B. distachyon as a model plant for biomass conversion of energy crops has to be specifically tested based on applied processing parameters and biomass tissue type.     

Pollen,ovules, and pollination in pea: Success,failure, and resilience in heat

Field pea (Pisum sativum), a major grain legume crop, is autogamous and adapted to temperate climates. The objectives of this study were to investigate effects of high temperature stress on stamen chemical composition, anther dehiscence, pollen viability, pollen interactions with pistil and ovules, and ovule growth and viability. Two cultivars (“CDC Golden” and “CDC Sage”) were exposed to 24/18°C (day/night) continually or to 35/18°C for 4 or 7 days. Heat stress altered stamen chemical composition, with lipid composition of “CDC Sage” being more stable compared with “CDC Golden.” Heat stress reduced pollen viability and the proportion of ovules that received a pollen tube. After 4 days at 35°C, pollen viability in flower buds decreased in “CDC Golden,” but not in “CDC Sage.” After 7 days, partial to full failure of anthers to dehisce resulted in subnormal pollen loads on stigmas. Although growth (ovule size) of fertilized ovules was stimulated by 35°C, heat stress tended to decrease ovule viability. Pollen appears susceptible to stress, but not many grains are needed for successful fertilization. Ovule fertilization and embryos are less susceptible to heat, but further research is warranted to link the exact degree of resilience to stress intensity.