Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Harmonization is more important than experience—results of the first Nordic–Baltic diatom intercalibration exercise 2007 (stream monitoring)

The goal of this study was a harmonization of diatom identification and counting among diatomists from the Scandinavian and Baltic countries to improve the comparison of diatom studies in this geographical area. An analysis of the results of 25 diatomists following the European standard EN 14407 during an intercalibration exercise showed that a high similarity was achieved by harmonization and not because of a long experience with diatoms. Sources of error were wrong calibration scales, overlooking of small taxa, especially small Navicula s.l., misidentifications (Eunotia rhomboidea was mistaken for Eunotia incisa) and unclear separation between certain taxa in the identification literature. The latter was discussed during a workshop with focus on the Achnanthes minutissima group, the separation of Fragilaria capucina var. gracilis from F. capucina var. rumpens, and Nitzschia palea var. palea from N. palea var. debilis. The exercise showed also that the Swedish standard diatom method tested here worked fine with acceptable error for the indices IPS (Indice de Polluo-sensibilité Spécifique) and ACID (ACidity Index for Diatoms) when diatomists with a low similarity (Bray–Curtis <60%) with the auditor in at least one of the samples are excluded.     

New method and criteria for national assessments of threatened habitat types

Consistent information on threatened habitat types is needed for land use planning and for prioritizing conservation, management, and restoration actions. However, detailed background data for assessing extinction risks of habitat types exists only in few countries. We present a new, flexible procedure for assigning habitat types into Red List Categories similar to those used for species by the World Conservation Union (IUCN). The procedure allows variation in the character or scale of assessment units and it is applicable even with incomplete data. The assessment protocol consists of two primary criteria: the change in the quantity and the change in the quality of the habitat type. The criteria are analyzed by expert groups with a transparent and repeatable stepwise procedure. The quantitative and qualitative changes in habitat types over the last 50 years serve as a starting point for the assessment, and the status is adjusted by assessing sub-criteria that address earlier changes, predicted future change, and the overall commonness or rarity of the habitat type. We also report the main results of the first assessment of threatened habitat types in Finland, and illustrate the application of the criteria by two case studies.     

Local and average gloss from flat-faced sodium chloride tablets

The purpose of this study was to detect local gloss and surface structure changes of sodium chloride tablets. The changes in surface structure were reflected by gloss variation, which was measured using a diffractive optical element-based gloss-meter (DOG). By scanning a surface area, we constructed a 2-dimensional gloss map that characterized the tablet’s surface structure. The gloss variation results were compared with scanning electron microscopy (SEM) images and average surface roughness values that were measured by conventional diamond stylus profilometry. The profilometry data showed a decrease in tablet surface roughness as a function of compression force. In general, a smoother surface contributes to higher average gloss values. The average gloss values for this material, in contrast, showed a decrease as a function of the compression force. The sequence of particle fragmentation and deformation together with crack formation in sodium chloride particles resulted in a loss of gloss for single sodium chloride particles at the tablet surfaces, which could be detected by the DOG. These results were supported by the SEM images. The results show that detailed information regarding tablets’ surface structure changes can be obtained by detection of local gloss variation and average gloss. Published: January 13, 2006     

Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide

Ligusticum chuanxiong and Angelica sinensis have been widely used in traditional Chinese medicine to treat some pathological settings such as atherosclerosis and hypertension. We determined the protective effect of the extract of Ligusticum chuanxiong and Angelica sinensis (ELCAS) on human umbilical vein endothelial cells (ECV304) damage induced by hydrogen peroxide. ECV304 cells were pre-treated with ELCAS and exposed to 5 mM hydrogen peroxide. The results show that ELCAS dose- and time-dependently protected ECV304 cells against hydrogen peroxide damage and suppressed the production of reactive oxygen species (ROS). The decrement of ROS may be associated with increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Western blot analysis revealed that ELCAS significantly increased the phosphorylation of ERK and promoted eNOS expression. These observations indicate that ELCAS protected ECV304 cells against hydrogen peroxide damage by enhancing the antioxidative ability, activating ERK and eNOS signaling pathway. Our data also provide new evidence of Ligusticum chuanxiong and Angelica sinensis in preventing both cardiovascular and cerebrovascular diseases.     

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD⁺ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD⁺ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 × 10^?14 mol NADH or NAD⁺ per assay.     

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases.

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital-induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well-defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48-h treatment of the hepatocytes with 2-aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose-dependent increases in both basal and phenobarbital-induced CYP2A5 catalytic activity (assayed as coumarin 7-hydroxylation), the maximal induction being 60-fold greater than the control value upon cotreatment with 1.5 mM phenobarbital and 10 mM 2-aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O-deethylase) and CYP1A1/2 (ethoxyresorufin O-deethylase) activities were blocked by 2-aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K-252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital-induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.     

Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.     

Biotic interactions improve prediction of boreal bird distributions at macro-scales

Aim The role of biotic interactions in influencing species distributions at macro‐scales remains poorly understood. Here we test whether predictions of distributions for four boreal owl species at two macro‐scales (10 × 10 km and 40 × 40 km grid resolutions) are improved by incorporating interactions with woodpeckers into climate envelope models. Location Finland, northern Europe. Methods Distribution data for four owl and six woodpecker species, along with data for six land cover and three climatic variables, were collated from 2861 10 × 10 km grid cells. Generalized additive models were calibrated using a 50% random sample of the species data from western Finland, and by repeating this procedure 20 times for each of the four owl species. Models were fitted using three sets of explanatory variables: (1) climate only; (2) climate and land cover; and (3) climate, land cover and two woodpecker interaction variables. Models were evaluated using three approaches: (1) examination of explained deviance; (2) four‐fold cross‐validation using the model calibration data; and (3) comparison of predicted and observed values for independent grid cells in eastern Finland. The model accuracy for approaches (2) and (3) was measured using the area under the curve of a receiver operating characteristic plot. Results At 10‐km resolution, inclusion of the distribution of woodpeckers as a predictor variable significantly improved the explanatory power, cross‐validation statistics and the predictive accuracy of the models. Inclusion of land cover led to similar improvements at 10‐km resolution, although these improvements were less apparent at 40‐km resolution for both land cover and biotic interactions. Main conclusions Predictions of species distributions at macro‐scales may be significantly improved by incorporating biotic interactions and land cover variables into models. Our results are important for models used to predict the impacts of climate change, and emphasize the need for comprehensive evaluation of the reliability of species–climate impact models.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.