Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.     

A targeted proteomics-based pipeline for verification of biomarkers in plasma

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Identification of Staphylococcus from French dry sausage

Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S. warneri and S. saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S. saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate. hydrolysis of tri-olein.     

Identification and field testing of floral odorants that attract the rove beetle <Emphasis Type="Italic">Pelecomalium testaceum</Emphasis> (Mannerheim) to skunk cabbage, <Emphasis Type="Italic">Lysichiton americanus</Emphasis> (L.)

Western skunk cabbage, Lysichiton americanus (Araceae), is pollinated mainly by the rove beetle Pelecomalium testaceum (Staphylinidae). Our objective was to determine the floral semiochemical(s) of L. americanus that attract(s) P. testaceum. Porapak Q headspace volatile extracts of L. americanus inflorescences were analyzed by gas chromatographic–electroantennographic detection (GC–EAD) and GC–mass spectrometry. In GC–EAD analyses, three floral odorants [(E)-4 nonene, (E)-5-undecene, indole] elicited consistent responses from the antennae of female P. testaceum. In field experiments, traps baited with a blend of these three components (“3-CB”) captured significantly more P. testaceum than unbaited control traps. Traps baited with the 3-CB, the two hydrocarbons, or indole, each captured significantly more beetles than unbaited control traps, indicating redundancy in the semiochemical blend. Moreover, traps baited with indole captured significantly more beetles than traps baited with either the 3-CB, or the hydrocarbons, indicating that indole is a key floral attractant for P. testaceum. Many necrophilous and coprophilous insects respond to indole in search of carrion or feces, but P. testaceum has never been associated with these types of resources. Both electrophysiological and behavioral responses of P. testaceum to two hydrocarbon semiochemicals, which are not signature odorants of carrion or feces, may indicate that the beetles recognize the odor of L. americanus as an honest signal, seek and pollinate its inflorescences, and get rewarded with pollen and on-plant mating opportunities.     

Plant cell culture technology in the cosmetics and food industries: current state and future trends

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.

     

Ustilago maydisMating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bölker, M., and Kahmann, R. 1996.Ustilago maydismating hyphae orient their growth toward pheromone sources.Fungal Genetics and Biology20,299–312. When small drops ofUstilago maydissporidia were placed 100–200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example,a2b2mating hyphae grew towarda1b1anda1b2mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with thea2allele began elongating before sporidia with thea1allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous atbbut not ataformed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, thea2a2b1b2strain formed filaments more quickly than thea1a1b1b2strain. Taken together, these results suggest that thea2pheromone diffuses less readily or is degraded more quickly than thea1pheromone.     

Plant species diversity and polyploidy in islands of natural vegetation isolated in extensive cultivated lands

Natural polyploidy is often related to a longer life span, vegetative reproduction and higher competitive ability. In this paper, we test the possibility that these characteristics may favour the survival of polyploid taxa under conditions of long-term habitat fragmentation. In islands of natural vegetation isolated in extensive vineyards located in the South of France and in a large neighbouring area of natural vegetation, plant species richness and the relative abundance of polyploid taxa were assessed according to island size, isolation and vegetation structure. High species richness was observed, with numerous species restricted to the islands, suggesting that these may constitute refugia. However, species richness was not related to island size or to degree of isolation except for the flora of the woody areas. A very positive effect of area fragmentation on plant richness was observed, which is probably attributable to relatively low species overlap among the islands. Particularly high species richness was observed in open areas, provided that these were not extensively colonized by shrubs which seem to be responsible for local extinction of many annual taxa. Polyploids, which comprised mostly perennial herbs and woody species, were predominant in all the islands and in the large reference area. In open habitats invaded by shrubby species, a higher relative frequency of polyploids was observed in islands than in the reference area. Moreover, polyploid taxa were present in a larger number of islands than the diploid taxa, which were often restricted to a single island, suggesting that, after a long period of isolation, the polyploids may still have a lower probability of extinction. Evidence was obtained from vegetation structure analysis that diploid and polyploid annual herbs were restricted to open habitats and were both eliminated by shrubby species. Conversely, the diploid perennial herbs were also significantly affected by shrub colonization whereas the polyploids were mostly present in shrubby areas. This suggests that the higher competitive ability of polyploid perennial herbs may constitute a critical factor responsible for their wider distribution over the islands. We report the implications of our findings on conservation strategies, more particularly for a Mediterranean flora.     

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.