An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.     

What is more important for invertebrate colonization in a stream with low-quality litter inputs: exposure time or leaf species?

The objective of this study was to evaluate the influences of detritus from the leaves of different species, and of exposure time on invertebrate colonization of leaves in a shaded Cerrado stream. We hypothesized that the exposure time is the main factor that influences the colonization of leaves by invertebrates. We used leaves of five tree species native to the Brazilian Cerrado: Protium heptaphyllum and Protium brasiliense (Burseraceae), Ocotea sp. (Lauraceae), Myrcia guyanensis (Myrtaceae), and Miconia chartacea (Melastomataceae), which are characterized by their toughness and low-nutritional quality. Litter bags, each containing leaves from one species, were placed in a headwater stream and removed after 7, 15, 30, 60, 90, and 120 days. The dominant taxon was Chironomidae, which comprised ca. 52% of all organisms and ca. 20% of the total biomass. The taxonomic richness of colonizing organisms did not vary among the leaf species. However, the density and biomass of the associated organisms varied differently among the kinds of detritus during the course of the incubation. The collector-gatherers and shredders reached higher densities in the detritus that decomposed more rapidly (Ocotea sp. and M. guyanensis), principally in the more advanced stages of colonization. The collector-filterers reached higher densities in the detritus that decomposed more slowly (P. heptaphyllum, P. brasiliense, and M. chartacea), principally in the initial stages of incubation. A cluster analysis divided the detritus samples of different leaf species according to the exposure time (initial phase: up to 7 days; intermediate phase: 7–30 days; advanced phase: 30–120 days), suggesting some succession in invertebrate colonization, with differences in taxon composition (indicator taxa analysis). These results suggest that regardless of the leaf-detritus species, exposure time was the main factor that influenced the colonization process of aquatic invertebrates.     

Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca²⁺-dependent manner at the previously identified “calmodulin-binding site” of SK1. We also demonstrate that CIB1 functions as a Ca²⁺-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.     

Triterpenoid saponins from the stem bark of Caryocar villosum

Five triterpenoid saponins, caryocarosides II-22 (3), III-22 (4), II-23 (5), III-23 (6), and II-24 (7), have been isolated from the methanol extract of the stem bark of Caryocar villosum, along with two known saponins (1-2). The seven saponins are glucuronides of hederagenin (II) or bayogenin (III). Caryocaroside II-24 (7) is an unusual galloyl ester saponin acylated on the sugar chain attached to C-28, the 3-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl hederagenin-28-O-[2-O-galloyl-beta-D-glucopyranosyl] ester. The structures of the saponins were established on the basis of extensive NMR ((13)C, (1)H, COSY, TOCSY, HSQC, HMBC and ROESY) and ESI-MS studies. The cytotoxic activity of saponins 2 and 3 was evaluated in vitro against human keratinocytes. The DOPA-oxidase inhibition and the lipolytic activities were evaluated ex vivo using an explant of human adipose tissue.     

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.     

Cephalotes clypeatus Fabricius (Hymenoptera: Formicidae): nesting habits and occurrence in animal carcass

The ecological position of the family Formicidae in animal carcasses varies from predator, when feeding on eggs, larvae and pupae of some insects to necrophagous, when the ants feed on exudates or decomposing tissues. Ants are present in human corpses subject to forensic analyses and can also be used in estimation of the post-mortem interval (PMI). Cephalotes clypeatus Fabricius is exclusively arboricolous and occurs only in the American continent. During a field study conducted in the Campus of the Universidade Estadual de Campinas, in December 2003, a laboratory mouse carcass weighing 35,9 g was placed in an iron-mesh cage, which was adequate to collect adult ants. The carcass decomposed in four days. The total of 82 specimens of C. clypeatus was collected, in the first two days of exposure. They were observed feeding on exudates, tissues of the carcass, and on Diptera larvae occurring in the carcass. This species was observed nesting in hollow branches of Senna multijuga (Rich.) H.S. Irwin & Barneby (Caesalpinaceae), which was found one-meter far from the cage. Further investigation on the biology of this Cephalotini must be performed, in order to understand the role of this species in the utilization of animal carcasses, and in the entomological succession process as well. This is the first report of C. clypeatus in animal carcasses.     

5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition

NMDA antagonists derived from 5-phosphonomethyl-1,4-dihydroquinoxaline-2,3-dione (3a) are potent anticonvulsant agents, and display strong protective effects in the electroshock-induced convulsion assay in mice. Their preference for the human NMDAR 1A/2A over 1A/2B subunit composition was optimized, leading to (1RS,1'S)-PEAQX (9r), which shows a >100-fold selectivity.     

Passive acoustic density estimation of sperm whales in the Tongue of the Ocean,Bahamas

Long‐term passive acoustic monitoring of marine mammals on navy ranges provides the opportunity to better understand the potential impact of sonar on populations. The navy range in Tongue of the Ocean (TOTO), Bahamas contains extensive hydrophone arrays, potentially allowing estimation of the density of deep diving, vocally active species such as the sperm whale (Physeter macrocephalus). Previous visual surveys in TOTO have been of limited spatio–temporal coverage and resulted in only sporadic sightings of sperm whales, whereas passive acoustic observations suggest the species is present year round. However, until now the means of acoustically determining the specific number of individuals in each cluster has been limited. We used recently developed algorithms to identify the number of echolocating whales present during a 42 d study period. We screened a 297 h acoustic data set to determine the proportion of time animals were present; fifty 10 min samples during presence were analyzed to estimate the number of individuals vocalizing during each sample. These counts were combined with an independent estimate of the proportion of 10 min periods when tagged animals vocalize. The estimated average density was 0.16 whales/1,000 km² (CV 27%; 95% CI 0.095–0.264). The method is potentially applicable to other areas containing dense hydrophone arrays.     

TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.