Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION*

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase^WT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C₁₆-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase^WT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase^S508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C₁₆-ceramide. Taken together, these data support a crucial role for Ser⁵⁰⁸ in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.     

Molecular identification of prey in the stomach contents of Harp Seals (Pagophilus groenlandicus) using species-specific oligonucleotides

All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach.     

Sequence Alignment Reveals Possible MAPK Docking Motifs on HIV Proteins

Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs). MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.     

Phytotoxic Allelochemicals From Roots and Root Exudates of Leafy Spurge (Euphorbia esula L.)

Invasive plants are a widespread problem but the mechanisms used by these plants to become invasive are often unknown. The production of phytotoxic natural products by invasive weeds is one mechanism by which these species may become successful competitors. Here we conducted a bioactivity-driven fractionation of root extracts and exudates from the invasive plant leafy spurge (Euphorbia esula L.), and structurally characterized jatrophane diterpenes and ellagic acid derivatives. Ellagic acid derivatives and one of the jatrophane diterpenes, esulone A, have been previously reported from leafy spurge, but another of the jatrophane diterpenes, kasuinine B, has not. We show that these compounds are phytotoxic but affect plants in different ways, either inducing overall plant necrosis or reducing root branching and elongation.Key Words: phytotoxicity, allelochemicals, roots, root exudates, jatrophane diterpenes, kansuinine B, ellagic acid derivatives, leafy spurge, Euphorbia esula, Arabidopsis thaliana     

Ocean color observations of a surface water transport event: Implications for Pseudo-nitzschia on the Washington coast

A persistent patch of high biomass water, associated with the Juan de Fuca Eddy, is often observed in surface chlorophyll a images off the southwest coast of Vancouver Island, Canada. Outbreaks of toxic Pseudo-nitzschia spp. along the Washington, USA, coast are believed to correlate with the transport of waters from Juan de Fuca Eddy southward to Washington beaches. A time series of Sea-viewing Wide Field-of-view Sensor (SeaWiFS) satellite ocean color images from late May 1999 of coastal waters off Washington and Vancouver Island, processed for surface chlorophyll a concentration and spectral remote sensing reflectance, captured a transport event where water from the Juan de Fuca Eddy was transported onto the Washington shelf. Strong upwelling-favorable winds appeared to deform the patch over an 8-day period and move it southward into Washington coastal waters with surface velocities of approximately 8–16 km d⁻¹. SeaWiFS and sea surface temperature imagery showed the local phytoplankton response to wind-driven coastal upwelling restricted to a narrow (10–15 km) region along the Washington coast. Although we did not observe transport of high biomass water originating in the Juan de Fuca Eddy to Washington beaches in May 1999, transport of Pseudo-nitzschia cells could occur following a rapid shift to downwelling-favorable conditions. Tracking the trajectory of surface waters from the Juan de Fuca Eddy by remote sensing could be used to trigger conditional sampling for domoic acid along the Washington coast.     

WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing

WRN is unique among the five human RecQ DNA helicases in having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end joining. Metal-ion complex structures, active site mutations and activity assays reveal a nuclease mechanism mediated by two metal ions. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ-family replicative proofreading exonucleases, describing WRN-specific adaptations consistent with double-stranded DNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support DnaQ-like proofreading activities stimulated by Ku70/80, with implications for WRN functions in age-related pathologies and maintenance of genomic integrity.     

SNP-PHAGE – High throughput SNP discovery pipeline

Background  

Single nucleotide polymorphisms (SNPs) as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs or microsatellite) markers for fine mapping and association studies in several species. For SNP discovery from chromatogram data, several bioinformatics programs have to be combined to generate an analysis pipeline. Results have to be stored in a relational database to facilitate interrogation through queries or to generate data for further analyses such as determination of linkage disequilibrium and identification of common haplotypes. Although these tasks are routinely performed by several groups, an integrated open source SNP discovery pipeline that can be easily adapted by new groups interested in SNP marker development is currently unavailable.     

Body protein stores and isotopic indicators of N balance in female reindeer (Rangifer tarandus) during winter

We studied bred and unbred female reindeer (Rangifer tarandus tarandus) during 12 wk of winter when ambient temperatures were low and nitrogen (N) demand for fetal growth is highest in pregnant females. Animals were fed a complete pelleted diet ad lib. that contained 2.54% N in dry matter that was 80% +/- 2% (X +/- SD) digestible. Female reindeer lost 64% +/- 14% of body fat but gained 34% +/- 11% of lean mass from 10 wk prepartum to parturition. These changes were equivalent to average balances of -14.14 +/- 2.35 MJ d(-1) and 10 +/- 3 g N d(-1). Blood cells, serum, and urine declined in (15)N/(14)N in late winter as body protein was gained from the diet. Blood cells of newborn calves were more enriched in (15)N and (13)C than that of their mothers, indicating the deposition of fetal protein from maternal stores. To quantify pathways of N flow in reindeer, N balance was measured by confining animals to cages for 10 d at 4 wk from parturition. N balance was inversely related to (15)N/(14)N in urea-N but not related to (15)N/(14)N of blood cells, creatinine, and feces. The proportion of urea-N derived from body protein increased above 0.46 as N balance fell below -200 mg N kg(-0.75) d(-1). Proportions of urea-N from body protein were -0.01 +/- 0.21 in pregnant females before and after caging and were consistent with average body protein gain in winter. Storage of protein allows reindeer and caribou to tolerate diets that are low in N without impairing fetal development.