Plastic reproductive strategies in a clonal marine invertebrate

Plastic reproductive allocation may allow individuals to maximize their fitness when conditions vary. Mate availability is one condition that may determine the fitness of an individual's allocation strategy. Using a variety of methods, I detected evidence of plastic allocation to asexual (clonal) reproduction in response to mate availability in the brittle star Ophiactis savignyi. There were more mature individuals in populations in which both sexes were present, and clones from these populations had fewer clone-mates than clones from single-sex populations. Animals placed with mates in a field experiment divided less frequently than animals without a mate. These findings demonstrate that animals reduce their allocation to asexual reproduction when mates are present and when a loss of fecundity associated with cloning would decrease offspring production. This plasticity is probably adaptive because it maximizes sexual-reproductive potential when fertilization is more likely, but maximizes survival of the clone when mates are absent and gametes are unlikely to be converted to offspring.     

Evidence for S. cerevisiae fermentation in ancient wine

Saccharomyces cerevisiae is the principal yeast used in modern fermentation processes, including winemaking, breadmaking, and brewing. From residue present inside one of the earliest known wine jars from Egypt, we have extracted, amplified, and sequenced ribosomal DNA from S. cerevisiae. These results indicate that this organism was probably responsible for wine fermentation by at least 3150 B.C. This inference has major implications for the evolution of bread and beer yeasts, since it suggests that S. cerevisiae yeast, which occurs naturally on the surface bloom of grapes, was also used as an inoculum to ferment cereal products.     

A fluorescence-based,high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.     

A two-protein strategy for the functional loading of a cellular replicative DNA helicase

The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.     

Development of an attractant for the scarab pest Macrodactylus subspinosus (Coleoptera: Scarabaeidae)

Field trials were conducted over several seasons to determine the attractant most successful in luring adult rose chafers, Macrodactylus subspinosus (F.), to traps. During the first season, 20 compounds were compared with the standard lure, valeric acid + hexanoic acid + octyl butyrate (1:1:1). The two new standards establishedthat season were: valeric acid + 1-nonanol (1:1); and valeric acid + hexanoic acid + octyl butyrate + 1-nonanol (1:1:1:1). The following season, 36 compounds were evaluated, comparing them to the new standards. The performance of the standard binary lure valeric acid + 1-nonanol was improved when the alcohol 1-nonanol was replaced by its analog trans-2-nonenol and this was confirmed during the third season. At the same time, a second test was conducted with 29 new candidates, which were combined with valeric acid and compared with the standard: valeric acid + hexanoic acid + octyl butyrate + 1-nonanol (1:1:1:1). A control and the single compound alpha-ionone were included, resulting in the discovery of a new more powerful attractant, alpha-ionone. Testing of alpha-ionone continued the following season, at which time the initial leading candidate and new ones containing trans-2-nonenol were tested against the single attractant alpha-ionone and various combinations of it. A new five-component mixture of valeric acid, hexanoic acid, octyl butyrate, trans-2-nonenol, and alpha-ionone out performed all other lure combinations.     

Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum

We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-alpha-tubulin fusion protein. The GFP-alpha-tubulin incorporates into microtubules and, as reported by others [Neujahr et al., 1998], the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-alpha-tubulin incorporates into microtubules at a lower efficiency than does the endogenous alpha-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-alpha-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP-alpha-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-alpha-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

LTD₄ induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D? (LTD?) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD? on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD?. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT? cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD?, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT? receptor antagonist, blocked the LTD?-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD?-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD? and exogenous HB-EGF. These findings suggest LTD? induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.     

Divergence genetics analysis reveals historical population genetic processes leading to contrasting phylogeographic patterns in co-distributed species

Coalescent samplers are computational time machines for inferring the historical demographic genetic processes that have given rise to observable patterns of spatial genetic variation among contemporary populations. We have used traditional characterizations of population structure and coalescent‐based inferences about demographic processes to reconstruct the population histories of two co‐distributed marine species, the frilled dog whelk, Nucella lamellosa, and the bat star, Patiria miniata. Analyses of population structure were consistent with previous work in both species except that additional samples of N. lamellosa showed a larger regional genetic break on Vancouver Island (VI) rather than between the southern Alexander Archipelago as in P. miniata. Our understanding of the causes, rather than just the patterns, of spatial genetic variation was dramatically improved by coalescent analyses that emphasized variation in population divergence times. Overall, gene flow was greater in bat stars (planktonic development) than snails (benthic development) but spatially homogeneous within species. In both species, these large phylogeographic breaks corresponded to relatively ancient divergence times between populations rather than regionally restricted gene flow. Although only N. lamellosa shows a large break on VI, population separation times on VI are congruent between species, suggesting a similar response to late Pleistocene ice sheet expansion. The absence of a phylogeographic break in P. miniata on VI can be attributed to greater gene flow and larger effective population size in this species. Such insights put the relative significance of gene flow into a more comprehensive historical biogeographic context and have important implications for conservation and landscape genetic studies that emphasize the role of contemporary gene flow and connectivity in shaping patterns of population differentiation.