Recruitment of mitochondrial tRNA genes as auxiliary variability markers for both intra- and inter-species analysis: The paradigm of brown hare (Lepus europaeus)

We sequenced and analyzed the mitochondrial tRNA(Thr) and tRNA(Pro) genes from brown hare (Lepus europaeus) individuals of different geographic distribution and we investigated the role of various nucleotide substitutions that were detected. We compared these tRNAs with the respective available mitochondrial tRNA genes sequences within Lepus species and among mammals. The mutations that were detected represent specific and conserved polymorphisms that do not seem to affect the structural and functional features that are required for participation of tRNA molecules in mitochondrial protein synthesis. These changes however, possibly reflect on the evolutionary background of the species, which is based on the high intra-genomic variability and the evolutionary dynamic of the mitochondrial DNA. In an attempt to compare the phylogeny that is based on these specific tRNA genes with the phylogeny that is produced from sequencing data of the mitochondrial variable loop, we came up with results that indicate similar phylogeographic clusters. This observation implies that the tRNA mutations that were used for the present study have been well tolerated during evolution and they define an additional genetic and biochemical tag that can be used for such studies. Based on this notion and according to our results, we propose that mitochondrial tRNA genes can be used as valuable auxiliary molecular markers for contemporaneous and linked biochemical and genetic analyses.     

dGPredictor: Automated fragmentation method for metabolic reaction free energy prediction and de novo pathway design

Group contribution (GC) methods are conventionally used in thermodynamics analysis of metabolic pathways to estimate the standard Gibbs energy change (Δ_rG′^o) of enzymatic reactions from limited experimental measurements. However, these methods are limited by their dependence on manually curated groups and inability to capture stereochemical information, leading to low reaction coverage. Herein, we introduce an automated molecular fingerprint-based thermodynamic analysis tool called dGPredictor that enables the consideration of stereochemistry within metabolite structures and thus increases reaction coverage. dGPredictor has comparable prediction accuracy compared to existing GC methods and can capture Gibbs energy changes for isomerase and transferase reactions, which exhibit no overall group changes. We also demonstrate dGPredictor’s ability to predict the Gibbs energy change for novel reactions and seamless integration within de novo metabolic pathway design tools such as novoStoic for safeguarding against the inclusion of reaction steps with infeasible directionalities. To facilitate easy access to dGPredictor, we developed a graphical user interface to predict the standard Gibbs energy change for reactions at various pH and ionic strengths. The tool allows customized user input of known metabolites as KEGG IDs and novel metabolites as InChI strings (https://github.com/maranasgroup/dGPredictor).     

Synthesis, characterization, and DNA adsorption studies of ampholytic model conetworks based on cross-linked star copolymers

Five model conetworks based on cross-linked star ampholytic copolymers were synthesized by group transfer polymerization. The ampholytic copolymers were based on two hydrophilic monomers: the positively ionizable 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the negatively ionizable methacrylic acid (MAA). Ethylene glycol dimethacrylate was used as the cross-linker. These five ampholytic model conetworks were isomers based on equimolar DMAEMA-MAA copolymer stars of different architectures: heteroarm (two), star block (two), and statistical. The two networks based on the homopolymer stars were also synthesized. The MAA units were introduced via the polymerization of tetrahydropyranyl methacrylate and the acid hydrolysis of the latter after network formation. All the precursors to the (co)networks were characterized in terms of their molecular weights using gel permeation chromatography (GPC). The mass of the extractables from the (co)networks was measured and characterized in terms of molecular weight and composition using GPC and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The degrees of swelling (DS) of all the ampholytic conetworks were measured as a function of pH and were found to present a minimum at a pH value which was taken as the isoelectric point, pI. The DS and the pI values did not present a dependence on conetwork architecture. Finally, DNA adsorption studies onto the ampholyte conetworks indicated that DNA binding was governed by electrostatics.     

Nanoliposomes and their applications in food nanotechnology

Food nanotechnology involves the utilization of nanocarrier systems to stabilize the bioactive materials against a range of environmental and chemical changes as well as to improve their bioavailability. Nanoliposome technology presents exciting opportunities for food technologists in areas such as encapsulation and controlled release of food materials, as well as the enhanced bioavailability, stability, and shelf-life of sensitive ingredients. Liposomes and nanoliposomes have been used in the food industry to deliver flavors and nutrients and, more recently, have been investigated for their abilityto incorporate antimicrobials that could aid in the protection of food products against microbial contamination. In this paper, the main physicochemical properties of liposomes and nanoliposomes are described and some of the industrially applicable methods for their manufacture are reviewed. A summary of the application of nanoliposomes as carrier vehicles of nutrients, nutraceuticals, enzymes, food additives, and food antimicrobials is also presented.     

Crystallographic and computational studies on 4-phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide, an inhibitor of glycogen phosphorylase: comparison with alpha-D-glucose, N-acetyl-beta-D-glucopyranosylamine and N-benzoyl-N'-beta-D-glucopyranosyl urea binding

4-Phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide (glucosyltriazolylacetamide) has been studied in kinetic and crystallographic experiments with glycogen phosphorylase b (GPb), in an effort to utilize its potential as a lead for the design of potent antihyperglycaemic agents. Docking and molecular dynamics (MD) calculations have been used to monitor more closely the binding modes in operation and compare the results with experiment. Kinetic experiments in the direction of glycogen synthesis showed that glucosyltriazolylacetamide is a better inhibitor (K(i) = 0.18 mM) than the parent compound alpha-D-glucose (K(i) = 1.7 mM) or beta-D-glucose (K(i) = 7.4 mM) but less potent inhibitor than the lead compound N-acetyl-beta-D-glucopyranosylamine (K(i) = 32 microM). To elucidate the molecular basis underlying the inhibition of the newly identified compound, we determined the structure of GPb in complex with glucosyltriazolylacetamide at 100 K to 1.88 A resolution, and the structure of the compound in the free form. Glucosyltriazolylacetamide is accommodated in the catalytic site of the enzyme and the glucopyranose interacts in a manner similar to that observed in the GPb-alpha-D-glucose complex, while the substituent group in the beta-position of the C1 atom makes additional hydrogen bonding and van der Waals interactions to the protein. A bifurcated donor type hydrogen bonding involving O3H, N3, and N4 is seen as an important structural motif strengthening the binding of glucosyltriazolylacetamide with GP which necessitated change in the torsion about C8-N2 bond by about 62 degrees going from its free to the complex form with GPb. On binding to GP, glucosyltriazolylacetamide induces significant conformational changes in the vicinity of this site. Specifically, the 280s loop (residues 282-288) shifts 0.7 to 3.1 A (CA atoms) to accommodate glucosyltriazolylacetamide. These conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding compared with the lead compound. In the molecular modeling calculations, the GOLD docking runs with and without the crystallographic ordered cavity waters using the GoldScore scoring function, and without cavity waters using the ChemScore scoring function successfully reproduced the crystallographic binding conformation. However, the GLIDE docking calculations both with (GLIDE XP) and without (GLIDE SP and XP) the cavity water molecules were, impressively, further able to accurately reproduce the finer details of the GPb-glucosyltriazolylacetamide complex structure. The importance of cavity waters in flexible receptor MD calculations compared to "rigid" (docking) is analyzed and highlighted, while in the MD itself very little conformational flexibility of the glucosyltriazolylacetamide ligand was observed over the time scale of the simulations.     

Stress response and changes in amino acid requirements in Senegalese sole (<Emphasis Type="Italic">Solea senegalensis</Emphasis> Kaup 1858)

Fish in aquaculture are often exposed to various stressors that may change their ability to survive or limit growth. Amino acids are used for processes other than growth, including stress response. This study intended to analyse how repeated acute handling stress can affect growth and amino acid requirements in fish. Senegalese sole juveniles were weekly held in the air during 3 min (Handling) for 9 weeks; Control groups were left undisturbed. Growth and plasma levels of stress indicators and of free amino acids were assessed at the end of the experiment. Plasma cortisol and osmolality levels showed that fish in the Handling treatment were stressed, but growth was unaffected. Plasma amino acid concentrations indicate that their requirements in stressed fish were altered, which probably reflects the synthesis of proteins or other specific compounds related to stress response.     

PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by Yacoub et al. (Mol Cancer Ther 2008; 7:314-29) further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK(-/-) cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.     

Ontogeny of osmoregulation in postembryonic fish: a review

Salinity and its variations are among the key factors that affect survival, metabolism and distribution during the fish development. The successful establishment of a fish species in a given habitat depends on the ability of each developmental stage to cope with salinity through osmoregulation. It is well established that adult teleosts maintain their blood osmolality close to 300 mosM kg(-1) due to ion and water regulation effected at several sites: tegument, gut, branchial chambers, urinary organs. But fewer data are available in developing fish. We propose a review on the ontogeny of osmoregulation based on studies conducted in different species. Most teleost prelarvae are able to osmoregulate at hatch, and their ability increases in later stages. Before the occurrence of gills, the prelarval tegument where a high density of ionocytes (displaying high contents of Na+/K+-ATPase) is located appears temporarily as the main osmoregulatory site. Gills develop gradually during the prelarval stage along with the numerous ionocytes they support. The tegument and gill Na+/K+-ATPase activity varies ontogenetically. During the larval phase, the osmoregulatory function shifts from the skin to the gills, which become the main osmoregulatory site. The drinking rate normalized to body weight tends to decrease throughout development. The kidney and urinary bladder develop progressively during ontogeny and the capacity to produce hypotonic urine at low salinity increases accordingly. The development of the osmoregulatory functions is hormonally controlled. These events are inter-related and are correlated with changes in salinity tolerance, which often increases markedly at the metamorphic transition from larva to juvenile. In summary, the ability of ontogenetical stages of fish to tolerate salinity through osmoregulation relies on integumental ionocytes, then digestive tract development and drinking rate, developing branchial chambers and urinary organs. The physiological changes leading to variations in salinity tolerance are one of the main basis of the ontogenetical migrations or movements between habitats of different salinity regimes.     

Synthesis, characterization, and evaluation as transfection reagents of double-hydrophilic star copolymers: effect of star architecture

Five star polymers of the ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the nonionic hydrophilic methoxy hexa(ethylene glycol) methacrylate (HEGMA) were prepared by group transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as coupling agent. In particular, four isomeric star copolymers, one heteroarm, two star block and one statistical star, with 90% mol DMAEMA and 10% mol HEGMA, plus one star homopolymer of DMAEMA with degrees of polymerization of the arms equal to 20 were synthesized. The polymers were characterized in terms of their molar masses (MMs) and compositions using gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1H NMR) spectroscopy, respectively. The hydrodynamic diameters in water indicated some aggregation for all the star polymers except for the statistical copolymer star, while the pK values of the DMAEMA units were around 7 for all star polymers. All the star polymers were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. All four star copolymers showed decreased toxicity compared to that of the DMAEMA star homopolymer for the same amounts of star polymer tested. The star block copolymer with outer DMAEMA blocks exhibited the highest overall transfection efficiency, 11%, compared to that of all the star polymers examined in this study. This efficiency was the same as that of the commercially available transfection reagent SuperFect.     

Biocatalytic preparation of acylated derivatives of flavonoid glycosides enhances their antioxidant and antimicrobial activity

Enzymatic synthesis of acylated derivatives of a monosaccharidic flavonoid chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside as well as of a disaccharidic flavonoid chrysoeriol-7-[6'-O-acetyl-beta-D-allosyl-(1-->2)-beta-D-glucopyranoside], isolated from Greek endemic plants, was performed using an immobilized Candida antarctica lipase in non-toxic organic solvents. The influence of the reaction parameters such as the molar ratio of acyl donor to flavonoid, as well as the nature of the acyl donor, on the performance of the biocatalytic process was pointed out using the acylation of naringin as a model reaction. With vinyl laurate as acyl donor, the highest conversion was observed at relatively high molar ratio (>or=10), using acetone as solvent. Lipase exhibits specificity towards primary alcohol of the glucose moiety of both flavonoid glycosides. The introduction of an acyl group into glucosylated flavonoids significantly improved their antioxidant activity towards both LDL and serum model in vitro. Furthermore, the acylated derivative of disaccharidic flavonoid increased its antimicrobial activity against two Gram-positive bacteria.