The External RNA Controls Consortium: a progress report

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.     

Mercury and persistent organic pollutant concentrations in African fish eagles, marabou storks, and Nile tilapia in Uganda

The purpose of this research was to evaluate persistent organic pollutant (POP) and mercury concentrations in tissues of African fish eagles (Haliaeetus vocifer) and Nile tilapia (Oreochromis niloticus) from Lake Victoria near Entebbe and Lake Mburo, Uganda. Marabou stork (Leptoptilos crumeniferus) nestlings from urban Kampala (40 km from Entebbe) also were sampled for POPs and mercury. Total mercury was measured in the breast feathers of eight nestling and 10 adult African fish eagles from Lake Mburo, 10 nestling and five adult African fish eagles from Lake Victoria near Entebbe, and 20 nestling marabou storks from Kampala from June 2002 through January 2003. Mercury concentrations in all samples were below levels associated with adverse effects in similar species. Mercury concentrations were significantly higher in eagle adults and nestlings from Entebbe than in adults and nestlings from Lake Mburo (P< or =0.05). No significant differences (P> or =0.05) were found in mercury concentrations between sexes or between the entire fish eagle population sampled at Entebbe and marabou stork nestlings sampled at nearby Kampala. Plasma samples from the same birds were analyzed for 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane, aldrin, hexachlorocyclohexane (alpha-HCH), dieldrin, endrin, heptachlor and their metabolites, as well as total polychlorinated biphenyls (PCBs). Nile tilapia whole-body cross sections collected from Lake Mburo (n=3) and Lake Victoria near Entebbe (n=8) also were analyzed for these POPs and mercury. No samples contained POPs or PCBs at the limits of detection except for 4,4'-1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene in five adult eagle plasma samples (0.0026+/-0.0015 ppm wet weight) and five Nile tilapia samples (0.002+/-0.001 ppm wet weight) from Entebbe.     

In vitro differentiation and antigenic changes in human melanoma cell lines

Summary Malignant transformation of melanocytes may be associated with changes in the expression of HLA antigens and melanoma-associated antigens (MAA). To determine whether these changes reflect the differential expression of HLA antigens and MAA by melanocytes at different stages of differentiation, we have studied the effect of the reversible induction of differentiation by fibroblast interferon (interferon ) and/or 12-O-tetradecanoyl-phorbol 13-acetate (TPA) on the expression of HLA antigens and MAA by the melanoma cell lines DU-2, FO-1 and HO-1. The three melanoma cell lines differed in their sensitivity to the differentiating and antiproliferative activity of these two compounds and displayed an increased growth suppression and induction of differentiation, when incubated with the combination of TPA and interferon . Incubation of the three melanoma cell lines with interferon , TPA or their combination resulted in a differential modulation of the expression of membrane-bound high-molecular-mass melanoma-associated antigen, 115-kDa MAA, 100-kDa MAA, intercellular adhesion molecule 1, HLA class I antigens and gene products of the HLA-D region. Each melanoma cell line displayed a unique pattern of antigenic modulation when exposed to the two differentiating agents alone or in combination. No direct relationship was found between the effects of interferon and/or TPA on the growth and differentiation of the three melanoma cell lines and the expression of HLA antigens or the MAA evaluated in the present study. These findings argue against a direct role of any of the antigens tested in the reversible induction of human melanoma cell differentiation in the in vitro system.     

A BLOC-1 mutation screen reveals a novel BLOC1S3 mutation in Hermansky-Pudlak Syndrome type 8

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1-6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient's melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.     

Comparison of foliar terpenes between native and invasive Solidago gigantea

To test a defensive chemistry prediction of the Evolution of Increased Competitive Ability (EICA) hypothesis, Solidago gigantea plants from North American and European (invasive) populations were grown in a screen-enclosed garden. Terpenes from 80 seed grown (dried leaves) and 320 rhizome propagated (moist leaves) individuals were confirmed by GC/MS and quantified by GC-FID. Native seed grown plants were found to have significantly greater diterpene concentrations than their European counterparts; foliar sesquiterpenes did not differ. The occurrence of specific sesquiterpenes and diterpenes was homogeneous across the two seed sources suggesting these biochemical pathways remain unchanged. Leaves from native rhizome propagated plants also had significantly greater monoterpene and diterpene concentrations; again sesquiterpene levels did not differ. Rhizome propagated plants exhibited significant population differences in monoterpene and diterpene concentrations. These data support the defensive chemistry predictions of the EICA hypotheses but cannot discount the role of possible founder effects in the invasive range.     

Exercise Modulates Redox-Sensitive Small GTPase Activity in the Brain Microvasculature in a Model of Brain Metastasis Formation

Tumor cell extravasation into the brain requires passage through the blood-brain barrier (BBB). There is evidence that exercise can alter the oxidation status of the brain microvasculature and protect against tumor cell invasion into the brain, although the mechanisms are not well understood. In the current study, we focused on the role of microenvironment generated by exercise and metastasizing tumor cells at the levels of brain microvessels, influencing oxidative stress-mediated responses and activation of redox-sensitive small GTPases. Mature male mice were exercised for four weeks using a running wheel with the average voluntary running distance 9.0±0.3 km/day. Mice were then infused with 1.0×10⁶ D122 (murine Lewis lung carcinoma) cells into the brain microvasculature, and euthanized either 48 hours (in short-term studies) or 2–3 weeks (in long-term studies) post tumor cell administration. A significant increase in the level of reactive oxygen species was observed following 48 hours or 3 weeks of tumor cells growth, which was accompanied by a reduction in MnSOD expression in the exercised mice. Activation of the small GTPase Rho was negatively correlated with running distance in the tumor cell infused mice. Together, these data suggest that exercise may play a significant role during aggressive metastatic invasion, especially at higher intensities in pre-trained individuals.     

Novel tricyclic antagonists of the prostaglandin D2 receptor DP2 with efficacy in a murine model of allergic rhinitis

The synthesis of a series of tricyclic antagonists for the prostaglandin D₂ receptor DP2 (CRTH2) is disclosed. The activities of the compounds were evaluated in a human DP2 binding assay and a human whole blood eosinophil shape change assay. Potential metabolic liabilities of the compounds were addressed through in vitro CYP studies. The lead compound was demonstrated to have efficacy in a mouse model of allergic rhinitis following oral dosing.     

Amphibian chytrid fungus Batrachochytrium dendrobatidis in Cusuco National Park, Honduras

Amphibian population declines in Honduras have long been attributed to habitat degradation and pollution, but an increasing number of declines are now being observed from within the boundaries of national parks in pristine montane environments. The amphibian chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines and was recently documented in Honduras from samples collected in Pico Bonito National Park in 2003. This report now confirms Cusuco National Park, a protected cloud forest reserve with reported amphibian declines, to be the second known site of infection for Honduras. B. dendrobatidis infection was detected in 5 amphibian species: Craugastor rostralis, Duellmanohyla soralia, Lithobates maculata, Plectrohyla dasypus, and Ptychohyla hypomykter. D. soralia, P. dasypus, and P. hypomykter are listed as critically endangered in the IUCN Red List of Threatened Species and have severely fragmented or restricted distributions. Further investigations are necessary to determine whether observed infection levels indicate an active B. dendrobatidis epizootic with the potential to cause further population declines and extinction.     

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.     

Response of medium‐ and large‐sized terrestrial fauna to corridor restoration along the middle Sacramento River

A fundamental challenge in restoration ecology is to understand when species are expected to colonize newly created habitat. Determining this is important for assessing progress toward restoration goals and, more generally, for gaining insight into ecosystem functioning and dynamics. We studied this question as it relates to mid‐ to large‐sized terrestrial fauna in restored riparian habitats at the Sacramento River National Wildlife Refuge, in northern California. We used camera traps to document use of 16 riparian corridor sites of varying restoration age. Comparisons of species richness (diversity) and visitation frequency (activity) were made across different‐aged sites. We found that predator diversity and activity levels tended to be higher in restored forests than in remnant forests, and that they tended to be highest in young restored forests. This trend persisted when data from variable sampling periods were pooled, although significant differences occurred more often in wet and cold sampling periods. The trend did not always hold for the animal community at large (consisting of both predator and non‐predator species). We conclude that restoration age affects predator diversity and activity levels in restored and remnant floodplain forests, and that predator communities can establish soon after restoration. Our results suggest that restoring natural river processes that promote habitat regeneration may benefit mid‐ to large‐sized terrestrial predators that appear to mostly use early successional habitat.