A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.     

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30°C at a pH of 5.0.     

Seasonal variation in food availability and relative importance of dietary items in the Gambian epauletted fruit bat (Epomophorus gambianus)

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Identification of risk-related haplotypes with the use of multiple SNPs from nuclear families

Family-based association studies offer robustness to population stratification and can provide insight into maternally mediated and parent-of-origin effects. Usually, such studies investigate multiple markers covering a gene or chromosomal region of interest. We propose a simple and general method to test the association of a disease trait with multiple, possibly linked SNP markers and, subsequently, to nominate a set of “risk-haplotype-tagging alleles.” Our test, the max_Z² test, uses only the genotypes of affected individuals and their parents without requiring the user to either know or assign haplotypes and their phases. It also accommodates sporadically missing SNP data. In the spirit of the pedigree disequilibrium test, our procedure requires only a vector of differences with expected value 0 under the null hypothesis. To enhance power against a range of alternatives when genotype data are complete, we also consider a method for combining multiple tests; here, we combine max_Z² and Hotelling’s T². To facilitate discovery of risk-related haplotypes, we develop a simple procedure for nominating risk-haplotype-tagging alleles. Our procedures can also be used to study maternally mediated genetic effects and to explore imprinting. We compare the statistical power of several competing testing procedures through simulation studies of case-parents triads, whose diplotypes are simulated on the basis of draws from the HapMap-based known haplotypes of four genes. In our simulations, the max_Z² test and the max_TDT (transmission/disequilibrium test) proposed by McIntyre et al. perform almost identically, but max_Z², unlike max_TDT, extends directly to the investigation of maternal effects. As an illustration, we reanalyze data from a previously reported orofacial cleft study, to now investigate both fetal and maternal effects of the IRF6 gene.     

Synthesis, X-ray structure and antimycobacterial activity of silver complexes with alpha-hydroxycarboxylic acids

In this paper, synthesis, characterization and antimycobacterial properties of a new water-soluble complex identified as silver-mandelate are described. Elemental and thermal analyses are consistent with the formula [Ag(C(6)H(5)C(OH)COO)](n). The polymeric structure was determined by single X-ray diffraction and the two-dimensional structure is based on the bis(carboxylate-O,O') dimer [Ag-O, 2.237(3), 2.222(3) Angstrom]. The structure is extended along both the b and c axes through two oxygen atoms of a bidentate alpha-hydroxyl-carboxylate residue [Ag-OH(hydroxyl), 2.477(3) Angstrom; Ag-O(carboxylate), 2.502(3) Angstrom; O-Ag-O, 63.94(9) degrees]. A strong d(10)-d(10) interaction was observed between two silver atoms. The Ag - Ag distance is 2.8307(15) Angstrom. The NMR (13)C spectrum in D(2)O shows that coordination of the ligand to Ag(I) occurs through the carboxylate group in solution. Potentiometric titration shows that only species with a molar metal:ligand ratio of 2:2 are formed in aqueous solution. The mandelate complex and the silver-glycolate, silver-malate and silver-hydrogen-tartarate complexes were tested against three types of mycobacteria, Mycobacterium avium, Mycobacterium tuberculosis and Mycobacterium kansasii, and their minimal inhibitory concentration (MIC) values were determined. The results show that the four complexes are potential candidates for antiseptic or disinfectant drugs for discharged secretions of patients affected with tuberculosis.     

Reproductive traits of two species of suckermouth armored catfishes (Siluriformes: Loricariidae) from a coastal drainage in the southern limits of the Atlantic Forest,Brazil

Environmental Biology of Fishes - The reproductive traits of two loricariid catfishes, Ancistrus multispinis and Pareiorhaphis hypselurus, from tributaries of the Mampituba river basin, southern...     

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats.     

SSR marker diversity of soybean aphid resistance sources in North America.

The soybean aphid (Aphis glycines Matsumura) has become a major pest of soybean in North America since 2000. Seven aphid resistance sources, PI 71506, Dowling, Jackson, PI 567541B, PI 567598B, PI 567543C, and PI 567597C, have been identified. Knowledge of genetic relationships among these sources and their ancestral parents will help breeders develop new cultivars with different resistance genes. The objective of this research was to examine the genetic relationships among these resistance sources. Sixty-one lines were tested with 86 simple sequence repeat (SSR) markers from 20 linkage groups. Non-hierarchical (VARCLUS) and hierarchical (Ward's) clustering and multidimensional scaling (MDS) were used to determine relationships among the 61 lines. Two hundred and sixty-two alleles of the 86 SSR loci were detected with a mean polymorphism information content of 0.36. The 61 lines were grouped into 4 clusters by both clustering methods and the MDS results consistently corresponded to the assigned clusters. The 7 resistance sources were clustered into 3 different groups corresponding to their geographical origins and known pedigree information, indicating genetic differences among these sources. The largest variation was found among individuals within different clusters by analysis of molecular variance.     

Parasitism on medfly by Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in different guava cultivars

The parasitism efficiency of the Braconidae wasp, Diachasmimorpha longicaudata (Ashmead), was checked on four guava cultivars (Paluma, Sassaoca, Pedro Sato and Kumagai) infested with larvae of medfly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Five blocks of eight fruits, each with two fruits of each cultivar, were put inside C. capitata adult cages, during 2h for oviposition, and a week later, when the larvae inside guavas were developed, the fruits were exposed to parasitoids for 24h. The mean fruit weight, larvae mortality, number of pupae, percentage of medfly and parasitoid emergence were evaluated. There was not statistical difference among cultivars to weight, larvae mortality, number of pupae e emergence of medfly. The percentage of parasitism was higher in Pedro Sato cultivar (19.8%) compared with Kumagai cultivar (2.9%), but it was statistically similar to the other cultivars.     

CD16 (FcRγIII) as a potential marker of osteoclast precursors in psoriatic arthritis

Introduction  

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis characterized by bone erosion mediated by osteoclasts (OC). Our previous studies showed an elevated frequency of OC precursors (OCP) in PsA patients. Here, we examined if OC arise from CD16-positive monocytes in PsA.