Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.     

High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae

Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets. To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge. To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae. Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves. This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult. These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant. Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress. As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells. Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics.     

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.     

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.     

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.     

Regulation of T lymphocyte metabolism

Upon stimulation, lymphocytes develop from small resting cells into highly proliferative and secretory cells. Although a great deal of study has focused on the genetic program induced by Ag receptor signals, lymphocytes must also regulate their metabolic function to meet the energetic demands of activation. In this review, we discuss the changes in cellular metabolism that accompany lymphocyte activation, with a particular emphasis on glucose metabolism, a major source of both energy and biosynthetic building blocks. We will also cover the signaling pathways that positively and negatively regulate these changes to maintain metabolic homeostasis in cells that are rapidly growing, dividing, and differentiating.     

Substance P augments Borrelia burgdorferi-induced prostaglandin E2 production by murine microglia

Substance P is a ubiquitous CNS neuropeptide and has recently been demonstrated to augment immune cell function during inflammatory events. Central to the ability of substance P to modulate immune cell function is the interaction of substance P with the substance P neurokinin-1 receptor expressed by a variety of immune cells, including microglia. CNS involvement during Lyme disease can occur when Borrelia burgdorferi, the causative agent of Lyme disease, gains access to the CNS. In the present study, we demonstrate that substance P augments B. burgdorferi-induced expression of mRNA encoding COX-2 and subsequent secretion of PGE(2) by cultured, murine microglia. Furthermore, this effect is associated with the ability of substance P to enhance B. burgdorferi-induced NF-kappa B activation, as demonstrated by increased nuclear localization of the p65 (RelA) subunit of NF-kappa B in these cells. Interestingly, we demonstrate that substance P augments B. burgdorferi-induced expression of mRNA encoding two PGE(2) receptors, E-prostanoid receptor subtypes 2 and 4, as well as each receptor protein. In addition, these effects are mediated via interactions between substance P and its high affinity receptor, as evidenced by the absence of augmented PGE(2) synthesis in the presence of a specific neurokinin-1 receptor antagonist or in cells genetically deficient in the expression of these receptors. Taken together, the present demonstration that substance P can exacerbate B. burgdorferi-induced inflammatory responses in microglia in vitro may indicate a role for this neuropeptide in the development of CNS inflammation observed during human neuroborreliosis.     

Epistatic interaction between two nonstructural loci on chromosomes 7 and 3 influences hepatic lipase activity in BSB mice

BSB mice exhibit a wide range of obesity despite being produced by a backcross of lean C57BL/6J (B) x lean Mus spretus (SPRET/Pt) F1 animals x B. Previous linkage studies identified a quantitative trait locus (QTL) on mouse chromosome 7 with coincident peaks for hepatic lipase activity, obesity, and plasma cholesterol. However, these mice were not analyzed for gene x gene epistasis. Hepatic lipase activity is correlated with obesity and plasma cholesterol levels. In this study, we identified QTLs for plasma hepatic lipase activity with three statistical mapping methods: maximum likelihood interval mapping, Bayesian nonepistatic mapping, and Bayesian epistatic mapping. Bayesian epistatic mapping detected not only the QTL on chromosome 7 but also an additional QTL on chromosome 3, which has a weak main effect but a strong interaction with chromosome 7. SPRET/Pt alleles of the QTL on each chromosome promote hepatic lipase activity. The proportion of phenotypic variance explained by the epistatic effect is higher than that explained by the main effect of the QTL on chromosome 7.     

Salmonid Opsin Sequences Undergo Positive Selection and Indicate an Alternate Evolutionary Relationship in Oncorhynchus

Positive selection can be demonstrated by statistical analysis when non-synonymous nucleotide substitutions occur more frequently than synonymous substitutions (dN>dS). This pattern of sequence evolution has been observed in the rhodopsin gene of cichlids. Mutations in opsin genes resulting in amino acid (AA) replacement appear to be associated with the evolution of specific color patterns and the evolution of courtship behaviors. Within fish, AA replacements in opsin proteins have improved vision at great depths and have occurred in deep-sea species. Salmonids experience diverse photic environments during their life history. Furthermore, sexual selection has resulted in species-specific male and female coloration during spawning. To look for evidence of positive selection in salmonid opsins, we sequenced the RH1, RH2, LWS, SWS1, and SWS2 genes from six Pacific salmon species as well as the Atlantic salmon. These salmonids include landlocked and migratory species and species that vary in their coloration during spawning. In each opsin gene comparison from all species sampled, traditional dN:dS analysis did not indicate positive selection. However, the more sensitive Creevey–McInerney statistical analysis indicates that RH1 and RH2 experienced positive selection early in the evolution and speciation of salmonids.     

Ectoparasites of gray squirrels in two different habitats and screening of selected ectoparasites for bartonellae

Gray squirrels, Sciurus carolinensis, were livetrapped in 2 different habitat types, woodland (67 squirrels) and parkland (53 squirrels), in southeastern Georgia. Ectoparasites were recovered from anesthetized squirrels and compared between hosts from the 2 habitats. Because of the absence of low vegetation in parkland habitats, it was hypothesized that the ectoparasite fauna, especially ticks and chiggers, would be more diverse on woodland squirrels. The results were generally in agreement with this hypothesis. Seventeen species of ectoparasites were recovered from woodland squirrels, compared with 6 species from parkland squirrels. Five species of ticks and 3 species of chiggers parasitized the woodland squirrels compared with no ticks or chiggers on the parkland squirrels. Significantly higher infestation prevalences were recorded on woodland compared with parkland squirrels for the flea Orchopeas howardi, the tick Amblyomma americanum, and the mesostigmatid mite Androlaelaps fahrenholzi. The mean intensity for O. howardi also was significantly higher on woodland than on parkland squirrels. Because a new strain of Bartonella sp. was isolated recently from S. carolinensis in Georgia, selected ectoparasites from this study were screened for bartonellae by polymerase chain reaction (PCR). Some of the fleas and lice, but none of the mites tested, were PCR positive, suggesting that fleas, or lice, or both, might be vectors of bartonellae between squirrels. Six distinct strains of Bartonella sp. were detected, 2 in fleas and 4 in lice.