Phylo-VISTA: interactive visualization of multiple DNA sequence alignments

MOTIVATION: The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. RESULTS: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a framework based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. AVAILABILITY: Phylo-VISTA is available at http://www-gsd.lbl.gov/phylovista. It requires an Internet browser with Java Plug-in 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu     

LL5beta is a phosphatidylinositol (3,4,5)-trisphosphate sensor that can bind the cytoskeletal adaptor,gamma-filamin

We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5beta). The protein bound PtdIns(3,4,5)P(3) selectively in vitro. Strikingly, a substantial proportion of LL5beta became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P(3) levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta that could not bind PtdIns(3,4,5)P(3) were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P(3) levels, LL5beta was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns(3,4,5)P(3) levels, it became significantly plasma-membrane localized. The distribution of LL5beta is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P(3) indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P(3). In addition, LL5beta bound to the cytoskeletal adaptor, gamma-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma-filamin with GFP-LL5beta in the unidentified vesicles.     

Stable isotopes as indicators of altitudinal distributions and movements in an Ecuadorean hummingbird community

Altitudinal migration and dispersal is an important component of the life history of several temperate and tropical birds but remains poorly understood due to the limited success of mark and recapture techniques. Stable isotopes of hydrogen (deltaD) in rainfall, and to a lesser extent, carbon (delta13C) in plants are known to change with altitude and hence may provide the basis of a technique for tracking the altitudinal movements in birds and other wildlife. We investigated the potential for this technique by measuring delta13C, deltaD, and delta15N values in tail feathers of eight species of hummingbirds ( Phaethornis malaris, P. syrmatophorus, P. guy, Adelomyia melanogenys, Coeligena torquata, C. lutetiae, Metallura baroni, M. williami) along an altitudinal gradient (300-3,290 m asl) in the Andes Mountains of Ecuador. Feather delta13C and deltaD values were correlated and each changed significantly with elevation above 400 m. In general, we found good agreement between feather deltaD values and those predicted from a generalized relationship of precipitation and surface water deltaD with altitude. Similarly, feather delta13C values showed an enrichment of approximately 1.5 per thousand per 1,000 m over the linear portion of the elevational response. Stable-nitrogen isotope values were variable, and so did not provide useful information on elevation in birds, apart from trophic effects. Overall there appears to be good potential for using the (deltaD, delta13C) stable isotope approach to track altitudinal movements and to elucidate previously unrecognized patterns of life history variation in both temperate and tropical species that migrate across elevational isotopic gradients.     

Activation of phosphoinositide 3-kinase gamma by Ras

BACKGROUND: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation.RESULTS: We show that posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. We have found GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could we detect any evidence of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate.CONCLUSIONS: Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.     

High-performance liquid chromatography-mass spectrometry method for the determination of paroxetine in human plasma

A rapid and specific liquid chromatographic mass spectrometric (LC-MS-MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid-liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane-ethyl acetate. The standard curve was linear over a working range of 0.2-50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges -4.8 to -0.5% and -3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.     

Effects of dietary fat types on body fatness,leptin, and ARC leptin receptor,NPY, and AgRP mRNA expression

Some, but not all, fats are obesogenic. The aim of the present studies was to investigate the effects of changing type and amount of dietary fats on energy balance, fat deposition, leptin, and leptin-related neural peptides: leptin receptor, neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC), in C57Bl/6J mice. One week of feeding with a highly saturated fat diet resulted in ~50 and 20% reduction in hypothalamic arcuate NPY and AgRP mRNA levels, respectively, compared with a low-fat or an n-3 or n-6 polyunsaturated high-fat (PUFA) diet without change in energy intake, fat mass, plasma leptin levels, and leptin receptor or POMC mRNA. Similar neuropeptide results were seen at 7 wk, but by then epididymal fat mass and plasma leptin levels were significantly elevated in the saturated fat group compared with low-fat controls. In contrast, fat and leptin levels were reduced in the n-3 PUFA group compared with all other groups. At 7 wk, changing the saturated fat group to n-3 PUFA for 4 wk completely reversed the hyperleptinemia and increased adiposity and neuropeptide changes induced by saturated fat. Changing to a low-fat diet was much less effective. In summary, a highly saturated fat diet induces obesity without hyperphagia. A regulatory reduction in NPY and AgRP mRNA levels is unable to effectively counteract this obesogenic drive. Equally high fat diets emphasizing PUFAs may even protect against obesity.     

Morphological,cultural and pathogenic characteristics of Coniothyrium zuluense isolates from different plantation regions in South Africa

Coniothyrium canker caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus species in sub-tropical regions of South Africa. This disease is typified by necrotic bark lesions that coalesce to form large kino-impregnated cankers along the stems of trees. The strategy currently used to manage Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Thus, forest managers are interested in the genetic diversity of the pathogen and its potential to overcome disease resistance in planting stock. In this study, 344 isolates of C. zuluense from different plantation regions in South Africa were compared on the basis of colony colour, conidial morphology, growth characteristics on agar and pathogenicity to a susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were similar in size and shape. The fungus is slow growing in culture, which is indicative of its apparent biotrophic habit, with optimum growth observed at 30 °C. Isolates of C. zuluense displayed considerable variation in colony colour and pathogenicity in inoculation trials. Variation in morphology and pathogenicity amongst isolates suggests that C. zuluense has been present in South Africa for an extended period of time, or that it is changing rapidly due to strong directional selection pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p=0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotyping was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominant.