Refinement of the spatial structure of the gramicidin A ion channel]

The spatial structure of the gramicidin A (GA) transmembrane ion-channel was refined on the base of cross-peak volumes measured in NOESY spectra (mixing time tau m = 100 and 200 ms). The refinement methods included the comparison of experimental cross-peak volumes with those calculated for low-energy GA conformations, dynamic averaging of the low-energy conformation set and restrained energy minimization. Accuracy of the spatial structure determination was estimated by the penalty function Fr defined as a root mean square deviation of interproton distances corresponding to the calculated and experimental cross-peak volumes. As the initial conformation we used the right-handed pi 6,3 LD pi 6,3 LD helix established on the base of NMR data regardless of the cross-peak volumes. The conformation is in a good agreement with NOE cross-peak volumes (Fr 0.2 to 0.5 A depending on NOESY spectrum). For a number of NOEs formed by the side chain protons, distances errors were found as much as 0.5-2.0 A. Restrained energy minimization procedure had little further success. However some of these errors were eliminated by the change in torsional angle chi 2 of D-Leu12 and dynamic averaging of the Val7 side chain conformations. Apparently, majority of deviations of the calculated and experimental cross-peak volumes are due to the intramolecular mobility of GA and cannot be eliminated within the framework of rigid globule model. In summary the spatial structure of GA ion-channel can be thought as a set of low-energy conformations, differing by the side chain torsion angles chi 1 Val7 and chi 2 D-Leu4 and D-Leu10 and the orientation of the C-terminal ethanolamine group. Root mean square differences between the atomic coordinates of conformations are in the range of 0.3-0.8 A.     

Spending degrees of freedom in a poor economy: A case study of building a sightability model for moose in northeastern Minnesota

Sightability models are binary logistic-regression models used to estimate and adjust for visibility bias in wildlife-population surveys. Like many models in wildlife and ecology, sightability models are typically developed from small observational datasets with many candidate predictors. Aggressive model-selection methods are often employed to choose a best model for prediction and effect estimation, despite evidence that such methods can lead to overfitting (i.e., selected models may describe random error or noise rather than true predictor–response curves) and poor predictive ability. We used moose (Alces alces) sightability data from northeastern Minnesota (2005–2007) as a case study to illustrate an alternative approach, which we refer to as degrees-of-freedom (df) spending: sample-size guidelines are used to determine an acceptable level of model complexity and then a pre-specified model is fit to the data and used for inference. For comparison, we also constructed sightability models using Akaike's Information Criterion (AIC) step-down procedures and model averaging (based on a small set of models developed using df-spending guidelines). We used bootstrap procedures to mimic the process of model fitting and prediction, and to compute an index of overfitting, expected predictive accuracy, and model-selection uncertainty. The index of overfitting increased 13% when the number of candidate predictors was increased from three to eight and a best model was selected using step-down procedures. Likewise, model-selection uncertainty increased when the number of candidate predictors increased. Model averaging (based on R = 30 models with 1–3 predictors) effectively shrunk regression coefficients toward zero and produced similar estimates of precision to our 3-df pre-specified model. As such, model averaging may help to guard against overfitting when too many predictors are considered (relative to available sample size). The set of candidate models will influence the extent to which coefficients are shrunk toward zero, which has implications for how one might apply model averaging to problems traditionally approached using variable-selection methods. We often recommend the df-spending approach in our consulting work because it is easy to implement and it naturally forces investigators to think carefully about their models and predictors. Nonetheless, similar concepts should apply whether one is fitting 1 model or using multi-model inference. For example, model-building decisions should consider the effective sample size, and potential predictors should be screened (without looking at their relationship to the response) for missing data, narrow distributions, collinearity, potentially overly influential observations, and measurement errors (e.g., via logical error checks). © 2011 The Wildlife Society.     

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

 The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae. Received: 28 July 1998 / Accepted: 8 October 1998     

Dissection of GA 20-oxidase members affecting tomato morphology by RNAi-mediated silencing

GA 20-oxidase is a key enzyme involved in gibberellin (GA) biosynthesis. In tomato, the GA 20-oxidase gene family consists of three members: GA20ox1, GA20ox2, and GA20ox3. To investigate the roles of these three genes in regulating plant growth and development, we used RNA interference technology to generate three kinds of transgenic tomato plants with suppressed expression of each three individual genes. Suppression of GA20ox1 or GA20ox2 resulted in shorter stems, a decreased length of internodes, and small dark green leaves while plants with decreased expression of GA20ox3 had no visible changes on stems and leaves. The plants of the three transgenic lines can flower and set fruits normally, but the seeds from these plants germinated slower than that from the normal plants. Decreased levels of endogenous GAs were detected in the apex of the three transgenic lines. These results demonstrate that the three GA 20-oxidase genes play different roles in the control of plan vegetative growth, but show no effects on flower and fruit development.Equal contribution authors: J. Xiao and H. Li.     

Microcultures of lactic acid bacteria: characterization and selection of strains,optimization of nutrients and gallic acid concentration

Eighteen lactic acid bacteria (LAB) strains, isolated from coffee pulp silages were characterized according to both growth and gallic acid (GA) consumption. Prussian blue method was adapted to 96-well microplates to quantify GA in LAB microcultures. Normalized data of growth and GA consumption were used to characterize strains into four phenotypes. A number of 5 LAB strains showed more than 60% of tolerance to GA at 2 g/l; whereas at 10 g/l GA growth inhibition was detected to a different extent depending on each strain, although GA consumption was observed in seven studied strains (>60%). Lactobacillus plantarum L-08 was selected for further studies based on its capacity to degrade GA at 10 g/l (97%). MRS broth and GA concentrations were varied to study the effect on growth of LAB. Cell density and growth rate were optimized by response surface methodology and kinetic analysis. Maximum growth was attained after 7.5 h of cultivation, with a dilution factor of 1–1/2 and a GA concentration between 0.625 and 2.5 g/l. Results indicated that the main factor affecting LAB growth was GA concentration. The main contribution of this study was to propose a novel adaptation of a methodology to characterize and select LAB strains with detoxifying potential of simple phenolics based on GA consumption and tolerance. In addition, the methodology presented in this study integrated the well-known RSM with an experimental design based on successive dilutions.     

TOOLS FOR STUDYING THE CHLOROPLAST GENOME IN THE TRITICEAE (GRAMINEAE): AN ECORI MAP,A DIAGNOSTIC DELETION,AND SUPPORT FOR BROMUS AS AN OUTGROUP

Data on variation in the chloroplast genome can be used to estimate the phylogeny of the wheat tribe (Triticeae), but two tools have been needed—a restriction site map for at least one frequent-cutting enzyme, and a well-supported outgroup. This paper presents an EcoRI map, as well as maps for six other restriction enzymes, for two members of the tribe (Elytrigia repens and Secale cereale). These are compared to the maps for Bromus tectorum, a possible outgroup, and for Briza maxima, Lolium perenne, and Festuca rubra, other members of the same subfamily (Pooideae). A unique deletion of ca. 700 base pairs was discovered that provides a distinctive marker for the cytoplasm of plants with the S genome. The deletion also appears in Dasypyrum villosum (V genome). Cladograms based on restriction site data are unequivocal in the support for Bromus tectorum as a near relative of the Triticeae; the chloroplast phylogeny is very similar to the morphological cladogram for a comparable set of taxa.     

Isolation and characterization of fourteen novel microsatellite loci in the Hong Kong oyster, Crassostrea hongkongensis

Fourteen polymorphic microsatellite markers were isolated from the Hong Kong oyster, Crassostrea hongkongensis, with a partial genomic library enriched for tandem repeat sequences of (CA)₁₂, (GA)₁₂, (ATG)₆ and (TAGA)₄. Polymorphism of these loci was assessed in a sample of 48 wild unrelated individuals. The average allelic number of these polymorphic loci was 6.36 per locus, with a range of 4–16. The observed and expected heterozygosity varied from 0.208 to 0.729 (averaging 0.502) and from 0.193 to 0.789 (averaging 0.615), respectively. After Bonferroni correction (P > 0.0036), 11 of the 14 loci accorded with Hardy–Weinberg equilibrium, and the rest three were detected significant departure from HWE. Additionally, two loci (Ch103 and Ch104) showed significant linkage disequilibrium (P < 0.01). This is the first set of microsatellite loci developed in this species and would be useful for studies of population genetics, stock management and other relevant research in C. hongkongensis.     

Oral Iron Treatment Has a Positive Effect on Iron Metabolism in Elite Soccer Players

The purpose of this study was to assess the effects of oral iron supplementation on hematological and iron metabolism in elite soccer players. Thirty-five members of the Real Zaragoza SAD soccer team took part in this study: group A (GA, n = 24; Spanish Premier League) took an oral iron supplement of 80 mg day⁻¹ for 3 weeks, and group B (GB, n = 11; Spanish Third Division League) did not receive any supplementation. In GA, the parameters were measured before and after giving the iron supplements, while in GB, measurements were only made at the time of collecting the second set of data from GA. After supplementation, GA showed an increase in serum iron (SI) (P < 0.05), serum ferritin (Ftn) (P < 0.01), and transferrin saturation (Sat) (P < 0.01) with respect to the basal values. In addition, GA showed higher values of hematocrit (P < 0.01), mean corpuscular volume (P < 0.01), Ftn (P < 0.01), and Sat (P < 0.01) than GB. No significant differences were found in any other parameters. More specifically, a higher percentage of players had Ftn levels above upper limits in GA vs. GB (P < 0.05), and GB had a higher incidence of Ftn below lower limits with respect to subjects in GA (P < 0.01). Further, after treatment, 58.3% of GA had >800 mg of SI, while all players in GB presented levels below the lower limits. In conclusion, iron supplementation with 80 mg·day⁻¹ for 3 weeks, before the start of the soccer season, can be recommended for elite soccer players.     

Isolation and characterization of microsatellite loci in Tripterygion delaisi

We isolated 49 microsatellite loci from a genomic library of Tripterygion delaisi×anthosoma enriched for CA and GA repeats. Ten loci were screened in 30 individuals with high numbers of alleles per locus (averaging 15.5 ± 2.86) and observed heterozygosity (averaging 0.765 ± 0.052). No deviations from Hardy–Weinberg expectations were detected. These highly polymorphic markers will be useful in determining the spatial patterns of genetic diversity between and within subspecies of Tripterygion delaisi.     

A Total Evidence Phylogeny of the Arctoidea (Carnivora: Mammalia): Relationships Among Basal Taxa

A total evidence phylogenetic analysis was performed for 14 extant and 18 fossil caniform genera using a data matrix of 5.6 kbp of concatenated sequence data from six independent loci and 80 morphological characters from the cranium and dentition. Maximum parsimony analysis recovered a single most parsimonious cladogram (MPC). The topology of the extant taxa in the MPC agreed with previous molecular phylogenies. Phylogenetic positions for fossil taxa indicate that several taxa previously described as early members of extant families (e.g., Bathygale and Plesictis) are likely stem taxa at the base of the Arctoidea. Taxa in the “Paleomustelidae” were found to be paraphyletic, but a monophyletic Oligobuninae was recovered within this set of taxa. This clade was closely related to the extant genera Gulo and Martes, therefore, nested within the extant radiation of the family Mustelidae. This analysis provides a resolution to several discrepancies between phylogenies considering either fossil taxa or extant taxa separately, and provides a framework for incorporating fossil and extant taxa into comprehensive combined evidence analyses.     

Polymorphism of Microsatellite Markers in Papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

A set of 81 new microsatellite markers for Carica papaya L. previously identified by data mining using freely available sequence information from Genbank were tested for polymorphism using 30 germplasm accessions from the Papaya Germplasm Bank (PGM) at Embrapa Mandioca e Fruticultura Tropical (CNPMF) and 18 landraces. The data were used to estimate pairwise genetic distances between the genotypes. A neighbor-joining based dendrogram was used to define clusters and infer possible genetic structuring of the collection. Most microsatellites were polymorphic (73%), with an observed number of alleles per locus ranging from one to eleven. The levels of observed and expected heterozygosity for 51 polymorphic loci varied from 0.00 to 0.85 and from 0.08 to 0.82, averaging 0.19 and 0.59, respectively. Forty-four percent of microsatellites showed polymorphism information content (PIC) higher than 0.50. The compound microsatellites seem to be more informative than dinucleotide and trinucleotide repeats in average alleles per locus and PIC. Among dinucleotides, AG/TC or GA/CT repeat motifs exhibited more informativeness than TA/AT, GT/CA and TG/AC repeat motifs. The neighbor-joining analysis based on shared allele distance could differentiate all the papaya accessions and landraces as well as differences in their genetic structure. This set of markers will be useful for examining parentage, inbreeding and population structure in papaya.     

Is there a way to protect human immune cells against nanocytotoxicity?

The silver nanoparticles (AgNPs) prepared by chemical reduction with sodium hypophosphite as a reducing agent and sodium hexametaphosphate as a stabilising agent were highly cytotoxic against human cells (U-937 and HL-60). The aim of the study was to determine the impact of selected antioxidants: ascorbic acid (AA), gallic acid (GA), scavenger (trolox (TX)) and Ag⁺ chelator (N-acetylcysteine, NAC) on viability, modulation of inflammatory response and apoptosis index of cells treated by AgNPs. Selected protectants added individually or together affects the viability of cells treated by AgNPs (1?mg/L). The mixtures assuring the most efficient defense against AgNPs were: AgNPs?+?TX?+?AA, AgNPs?+?GA?+?AA, AgNPs?+?TX?+?GA?+?AA and AgNPs?+?TX?+?GA?+?AA?+?NAC which synergistically interact in the mixture. The greatest reduction in IL-6 and TNF-α levels was found for the mixture containing AgNPs?+?TX?+?GA?+?AA. Mixture of this composition exhibited also the strongest anti-apoptotic effect. Highly cytotoxic AgNPs may not damage human cells if cytoprotectants are present.     

Role of the gibberellin receptors GID1 during fruit‐set in Arabidopsis

Gibberellins (GAs) play a critical role in fruit‐set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit‐set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue‐specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C–RGL1 and GID1B–RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1–DELLA in the different GA‐dependent processes that occur upon fruit‐set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.     

Somatic embryogenesis,regeneration and <Emphasis Type="Italic">in vitro</Emphasis> production of glycyrrhizic acid from root cultures of <Emphasis Type="Italic">Taverniera cuneifolia</Emphasis> (Roth) Arn.

Taverniera cuneifolia (Roth) Arn. or Indian licorice is considered to be a substitute for Glycyrrhiza glabra L. owing to an equivalent content of glycyrrhizic acid (GA). GA recognized as the main active ingredient of T. cuneifolia, GA imparts several medicinal properties to these plants. However, research on this plant is scanty with no published record on tissue culture studies. Present study demonstrates a method for (1) induction and successful development of somatic embryos from the root culture, (2) regeneration of plants, and (3) GA production by root cultures of T. cuneifolia. Shoot initiation frequency of cultured roots ranged from 52.57% to 97.71%. Plant regeneration frequency (germination) from somatic embryos was 88.66% in 1/4-strength Murashige and Skoog (MS) medium supplemented with 2% sucrose, as against 68% in half 1/2-strength MS containing 2% sucrose. Glycyrrhizic acid of 1.32 mg/gm of dry weight was obtained in full-strength MS medium supplemented with 3% sucrose, through high-performance thin layer chromatography analysis with standard GA in root cultures. The present study provides an efficient method for the mass production of plants from a single mother plant as well as the potential root culture system to study the GA production pathways in T. cuneifolia.