Meeting Report from the Genomic Standards Consortium (GSC) Workshop 8

This report summarizes the proceedings of the 8th meeting of the Genomic Standards Consortium held at the Department of Energy Joint Genome Institute in Walnut Creek, CA, USA on September 9-11, 2009. This three-day workshop marked the maturing of Genomic Standards Consortium from an informal gathering of researchers interested in developing standards in the field of genomic and metagenomics to an established community with a defined governance mechanism, its own open access journal, and a family of established standards for describing genomes, metagenomes and marker studies (i.e. ribosomal RNA gene surveys). There will be increased efforts within the GSC to reach out to the wider scientific community via a range of new projects. Further information about the GSC and its activities can be found at http://gensc.org/.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Transmembrane signalling by the N-formyl peptide receptor in stably transfected fibroblasts

We investigated the requirement for N-formyl peptide receptor-mediated transmembrane signalling in transfected mouse fibroblasts that express the receptor. Stably transfected cells displayed specific binding for N-formyl-Met-Leu-[3H]Phe with a dissociation constant of 3 nM. The cells responded to ligand stimulation with mobilization of calcium from intracellular stores. Calcium mobilization was ligand dose-dependent (EC50 = 3 nM fMet-Leu-Phe) and could be inhibited by pertussis toxin treatment. These results provide the first demonstration that expression of the single-chain N-formyl peptide receptor in mouse fibroblasts is sufficient for mediating ligand-induced early transmembrane signalling events, which do not appear to require other neutrophil-specific cellular components.     

From global to regional and back again: common climate stressors of marine ecosystems relevant for adaptation across five ocean warming hotspots

Ocean warming ‘hotspots’ are regions characterized by above‐average temperature increases over recent years, for which there are significant consequences for both living marine resources and the societies that depend on them. As such, they represent early warning systems for understanding the impacts of marine climate change, and test‐beds for developing adaptation options for coping with those impacts. Here, we examine five hotspots off the coasts of eastern Australia, South Africa, Madagascar, India and Brazil. These particular hotspots have underpinned a large international partnership that is working towards improving community adaptation by characterizing, assessing and projecting the likely future of coastal‐marine food resources through the provision and sharing of knowledge. To inform this effort, we employ a high‐resolution global ocean model forced by Representative Concentration Pathway 8.5 and simulated to year 2099. In addition to the sea surface temperature, we analyse projected stratification, nutrient supply, primary production, anthropogenic CO₂‐driven ocean acidification, deoxygenation and ocean circulation. Our simulation finds that the temperature‐defined hotspots studied here will continue to experience warming but, with the exception of eastern Australia, may not remain the fastest warming ocean areas over the next century as the strongest warming is projected to occur in the subpolar and polar areas of the Northern Hemisphere. Additionally, we find that recent rapid change in SST is not necessarily an indicator that these areas are also hotspots of the other climatic stressors examined. However, a consistent facet of the hotspots studied here is that they are all strongly influenced by ocean circulation, which has already shown changes in the recent past and is projected to undergo further strong change into the future. In addition to the fast warming, change in local ocean circulation represents a distinct feature of present and future climate change impacting marine ecosystems in these areas.     

Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: formation of high-affinity ligand- receptor complexes in transient association with cytoskeleton

When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.     

Management procedures in a fishery based on highly variable stocks and with conflicting objectives: experiences in the South African pelagic fishery

The pelagic fishery in South Africa targets mainly anchovy, Engraulis capensis, and sardine, Sardinops sagax, both of which have varied substantially in abundance during the history of the fishery. Since 1988, there has been progress in this fishery towards the use of management procedures as the basis for determination of management regulations, where a management procedure is defined as a set of rules, derived by simulation and normally implemented for three to five years, specifying how the regulatory mechanism is set, the data collected for this purpose and how these data are to be analysed and used. Advantages of management procedures include formal consideration of uncertainty, the ability to choose decision rules based on their predicted medium-term consequences and a saving in workload compared with annual assessments.This paper discusses the lessons learned in application of management procedures and their precursors in this fishery. The high variability in abundance of the two stocks, the trend in their relative abundance, the substantial uncertainties in information, strong pressure to meet socio-economic goals and the conflicting objectives which arose between the directed anchovy and directed sardine fishery are identified as major problems in implementation of procedures and management of the resources. However, the use of management procedures is considered to have led to greatly improved communication with the industry and to substantial input by them into the management process. The procedures and the simulations upon which they were based also enabled consideration of the major sources of uncertainty in understanding of the resource dynamics and facilitated the development of procedures that were robust to them.It is argued that biological uncertainty greatly exacerbated the problems in application of the procedures but probably cannot be markedly reduced in the near future. Management procedures must be robust to likely variability and uncertainty. Of equal importance are identification and selection of achievable objectives, and allocation to the political decision makers and not to the scientists, of responsibility for determining acceptable trade-offs between conservation and socio-economic goals. Other issues, including the importance of long-term rights and allowance for flexibility in fishing practice, are also highlighted     

Observations on the Germ Aleurone of Barley. Phenol Oxidase and Peroxidase Activity

Strips of tissue containing the germ aleurone layer were removedfrom dry, harvest-ripe grains of barley (Hordeum vulgare L.)and incubated in buffered solutions of phenolic compounds, withand without the addition of hydrogen peroxide. Peroxidase ando-diphenol oxidase activity were found in the material releasedinto the incubation medium, and in the cytoplasm of the germaleurone cells. Peroxidase activity was located in the cellwalls and appeared to be high in the region where the germ aleuronecovering the embryonic axis merges into that which adheres tothe scutellum i.e. the region in which a row of germ aleuronecells becomes lignified following germination. Monophenol oxidaseactivity was not detected in the released enzymes or in theintact tissue. Although hydroquinone was oxidized in the cytoplasmof the germ aleurone tissue, unequivocal evidence of the presenceof laccase was not obtained. The oxidation of endogenous phenolicsubstances by phenol oxidases and peroxidases is discussed inrelation to anti-microbial defence mechanisms which appear tooperate in the germ aleurone during germination.Copyright 1994,1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, catechol oxidase, laccase, peroxidase, defence mechanisms, germination     

Immunocytochemical and biochemical studies of histamine in the retina of the turtle Pseudemys scripta

A combination of immunocytochemical and biochemical methods was used to study histamine in the turtle retina. Histamine-like immunoreactivity was localized within paraboloids of certain cone photoreceptors by use of two different antisera directed against histamine. Preincubation of eyecups in Ringer's containing 10 microM histamine selectively increased the immunoreactivity of these photoreceptor paraboloids. The present localization of histamine in paraboloids indicated that, although histamine is in photoreceptors of the turtle retina, it may play some metabolic or neuromodulatory role, and not function as a neurotransmitter.     

Regulation of neurotensin receptor function by the arachidonic acid-lipoxygenase pathway in prostate cancer PC3 cells

Neurotensin (NT) elevates leukotriene levels in animals and stimulates 5-HETE formation in prostate cancer PC3 cells. PC3 cell growth is stimulated by NT and inhibited by lipoxygenase (LOX) blockers. This led us to test LOX blockers (NDGA, MK886, ETYA, Rev5901, AA861 and others) for effects on NT binding and signaling. LOX blockers dramatically enhanced 125I-neurotensin binding to NT receptor NTR1 in PC3 cells, whereas they inhibited NT-induced inositol phosphate formation. These effects were indirect (binding to isolated membranes was unaffected), receptor-specific (binding to beta2-adrenergic, V1a-vasopressin, EGF and bombesin receptor was unaffected) and pathway-specific (cyclooxygenase inhibitors were inactive). NT receptor affinity was increased but receptor number and % internalization were unchanged. Also supporting the involvement of arachidonic acid metabolism in NTR1 regulation was the finding that inhibitors of PLA2 and DAG lipase enhanced NT binding. These findings suggest that NTR1 is regulated by specific feedback mechanism(s) involving lipid peroxidation and/or LOX-dependent processes.