MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Breast cancer risk and heterocyclic amines and effect modification by NAT gene polymorphisms

Breast cancer is the most common cancer in women in the United States and is a leading cause of cancer-related deaths (1). Recently, dietary heterocyclic amines (HCAs) have been proposed to be a risk factor for breast cancer (2). This study uses the data collected for a case-control study conducted at the M.D. Anderson Cancer Center to assess the association between breast cancer risk and HCAs {2-amino-1-methyl-6-phenylimidazole [4,5-b] pyridine (PhIP), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo [4,5-f] quinoxaline (DiMeIQx) and mutagenicity of HCAs} and to examine if this association is modified by genetic polymorphisms of N-acetyl transferases (NAT1/NAT2). The NAT1/2 genotype was determined using Taqman technology. HCAs were estimated by using a meat preparation questionnaire on meat type, cooking method, and doneness, combined with a quantitative HCA database. Three hundred and fifty patients with breast cancer attending the Diagnostic Radiology Clinic at M. D. Anderson Cancer Center and fulfilling the eligibility criteria were compared to three hundred and fifty patients attending the same clinic for benign breast lesions to answer these questions. Logistic regression models were used to control for known risk factors and showed no statistically significant association between breast cancer versus benign breast cancer lesions and dietary intake of heterocyclic amines. There was no clear difference in their effect after subgroup analyses in different acetylator strata of NAT1/2 and no statistical interactions were found between NAT1/2 genotypes and HCAs, suggesting no effect modification by NAT1/2 acetylator status. These results suggest the need for further research to analyze if these null associations were because of the benign breast lesions sharing the risk factors with breast cancer or any other factors which haven't been explored yet.^

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

PREPARATION AND CHARACTERIZATION OF FAD DEPENDENT NADPH CYTOCHROME P-450 REDUCTASE (FLAVOPROTEINS, APOFLAVOPROTEIN)

NADPH cytochrome P-450 reductase releases FMN and FAD upon dilution into slightly acidic potassium bromide. The flavins are released with positive cooperativity. Dithiothreitol protects the FAD dependent cytochrome c reductase activity against inactivation by free radicals. Behavior in potassium bromide is sensitive to changes in the pH. High performance hydroxylapatite resolved the FAD dependent reductase from holoreductase. For 96% FAD dependent reductase, the overall yield was 12%.^ High FAD dependence was matched by a low FAD content, with FAD/FMN as low as 0.015. There were three molecules of FMN for every four molecules of reductase. The aporeductase had negligible activity towards cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, oxygen and acetyl pyridine adenine dinucleotide phosphate. A four minute incubation in FAD reconstituted one half to all of the specific activity, per milligram protein, of untreated reductase, depending upon the substrate. After a two hour reconstitution, the reductase eluted from hydroxylapatite at the location of holoreductase. It had little flavin dependence, was equimolar in FMN and FAD, and had nearly the specific activity (per mole flavin) of untreated reductase.^ The lack of activity and the ability of FMN to also reconstitute suggest that the redox center of FAD is essential for catalysis, rather than for structure. Dependence upon FAD is consistent with existing hypotheses for the catalytic cycle of the reductase. ^

Modification of mutagen sensitivity by chemopreventive agents {\it in vitro\/}

The clinical application of chemopreventive agents is expected to prevent the appearance of cancer by arresting carcinogenesis or reversing it in the precancerous stages. The hypothesis of the present investigations was that chemopreventive agents (retinoids and antioxidant vitamins) may counteract the clastogenic effects of bleomycin in vitro in both lymphoblastoid cell lines and primary lymphocyte cultures and that a similar phenomenon can be detected in lymphocytes from individuals treated with 13-cis-retinoic acid. The efficacy of 13-cis-retinoic acid, n-(4-hydroxyphenyl)-retinamide, ascorbic acid, n-acetyl-l-cysteine, alpha-tocopherol, and alpha-tocopherol-acid succinate was tested against bleomycin-induced chromosomal breakage.^ The results provided direct evidence of the concentration-related protective effects of these agents against bleomycin-induced clastogenicity in cultures of human lymphoblastoid cell lines in vitro. Similar anticlastogenic protection was demonstrated with 13-cis-retinoic acid, ascorbic acid, n-acetyl-l-cysteine, and alpha-tocopherol-acid succinate in primary lymphocyte cultures in vitro. The in vitro anticlastogenic effect of 13-cis-retinoic acid was also demonstrated in lymphocyte cultures from peripheral blood samples from patients treated with this retinoid.^ An important consideration is that the concentrations used in the present investigations are comparable to those achieved in clinical situations.^ The in vitro anticlastogenic effect of these retinoids and antioxidants may constitute an important element of their chemopreventive properties. The results corroborate the hypothesis that these compounds may be effective in clinical chemoprevention trials. The bleomycin-assay may also be used as a short-term test to evaluate the antimutagenic effects of various agents. ^

Differential stress response and susceptibility to oxidative injury in alveolar macrophages from C57BL/6J and C3H/HeJ mice following oxidant exposure

Studies have demonstrated a variable response to ozone among individuals and animal species and strains. For instance, C57BL/6J mice have a greater inflammatory response to ozone exposure than C3H/HeJ mice. In these studies, I utilized these strain differences in an effort to derive a mechanistic explanation to the variable strain sensitivity to ozone exposure. Therefore, alveolar macrophages (AM) from C57BL/6J and C3H/HeJ mice were exposed in vitro to hydrogen peroxide ($\rm H\sb2O\sb2$), heat and acetyl ceramide or in vivo to ozone. Necrosis and DNA fragmentation in macrophages from the two murine strains were determined to assess cytotoxicity following these treatments. In addition, synthesis and expression of the stress proteins, stress protein 72 (SP72) and heme oxygenase (HO-1), were examined following treatments. The in vitro experiments were conducted to eliminate the possibility of in vivo confounders (i.e., differences in breathing rates in the two strains) and thus directly implicate some inherent difference between cells from the two murine strains. $\rm H\sb2O\sb2$ and heat caused greater cytotoxicity in AM from C57BL/6J than C3H/HeJ mice and DNA fragmentation was a particularly sensitive indicator of cell injury. Similarly, AM from C57BL/6J mice were more sensitive to ozone exposure than cells from C3H/HeJ mice. Exposure to either 1 or 0.4 ppm ozone caused greater cytotoxicity in macrophages from C57BL/6J mice compared to macrophages from C3H/HeJ mice. The increased sensitivity of AM to injury was associated with decreased synthesis and expression of stress proteins. AM from C57BL/6J mice synthesized and expressed significantly less stress proteins in response to heat and ozone than AM from C3H/HeJ mice. Heat treatment resulted in greater synthesis and expression of SP72. In addition, macrophages from C57BL/6J mice expressed lower amounts of HO-1 than macrophages from C3H/HeJ mice following 0.4 ppm ozone exposure. Therefore, AM from C57BL/6J mice are more susceptible to oxidative injury than AM from C3H/HeJ mice which might be due to differential expression of stress proteins in these cells. ^

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Modulation of BCL-X$\sb{\rm L}$ is a critical determinant of c-myc induced apoptosis

The fine balance between proliferation and apoptosis plays a primary role in carcinogenesis. Proto-oncogenes that induce both proliferation and apoptosis provide a powerful inbuilt system to inhibit clonal expansion of cells with high proliferation rates. This provides a restraint to the development of neoplasms. C-myc expressing cells undergo apoptosis in low serum by an unknown mechanism. Several lines of evidence suggested that c-myc induces apoptosis by a transcriptional mechanism. However, the target genes of this program have not been fully defined. Protein synthesis inhibitors induce apoptosis in c-myc over-expressing cells at high serum levels suggesting that inhibition of synthesis of a survival factor may induce apoptosis. We show that the expression of c-myc directly correlates with an increase in the level of a survival protein, bcl-$\rm x\sb{L},$ and a decrease in the pro-apoptotic protein, bax, at both the protein and mRNA level. Furthermore, a significant decrease of the bcl-$\rm x\sb{L}$ protein levels is observed under low serum conditions. In order to investigate the mechanism of regulation of bcl-$\rm x\sb{L}$ and bax by c-myc, the bcl-x and bax promoters were cloned, sequenced and shown to contain c-myc binding sites. The chloramephenicol acetyl transferase (CAT) reporter assay was used to demonstrate activation of the bcl-x promoter by increasing levels of c-myc when co-transfected in COS cells. The bax promoter was also shown to be transrepressed in c-myc expressing cells. The role of bcl-$\rm x\sb{L}$ in apoptosis regulation in c-myc cell lines in normal and low serum was then investigated. Cells lines expressing c-myc and bcl-$\rm x\sb{L}$ were generated and were shown to be resistant to apoptosis induction in low serum. Furthermore, cell lines expressing c-myc, anti-sense bcl-$\rm x\sb{L}$ and $\beta$-galactosidase demonstrated significantly enhanced rates of apoptosis in high serum compared to c-myc Rat 1a cells. These findings suggest that c-myc activates a survival program involving bcl-$\rm x\sb{L}$ upregulation and bax downregulation. However, this survival signal is reduced under low serum conditions by the relative downregulation of bcl-$\rm x\sb{L}$ allowing for apoptosis to proceed. These data also directly demonstrates that downregulation in the level of bcl-$\rm x\sb{L}$ associated with low serum conditions is a critical determinant of c-myc induced apoptosis. ^

Proceedings of the Twenty-First Annual Biochemical Engineering Symposium

The 21st Annual Biochemical Engineering Symposium was held at Colorado State University on April 20, 1991. The primary goals of this symposium series are to provide an opportunity for students to present and publish their research work and to promote informal discussions on biochemical engineering research. Contents High Density Fed-Batch Cultivation and Energy Metabolism of Bacillus thuringtensis; W.-M. Liu, V. Bihari, M. Starzak, and R.K. Bajpai Influences of Medium Composition and Cultivation Conditions on Recombinant Protein Production by Bacillus subtilis; K. Park, P.M. Linzmaier, and K.F. Reardon Characterization of a Foreign Gene Expression in a Recombinant T7 Expression System Infected with λ Phages; F. Miao and D.S. Kompala Simulation of an Enzymatic Membrane System with Forced Periodic Supply of Substrate; N. Nakaiwa, M. Yashima, L.T. Fan, and T. Ohmori Batch Extraction of Dilut Acids in a Hollow Fiber Module; D.G. O'Brien and C.E. Glatz Evaluation of a New Electrophoretic Device for Protein Purification; M.-J. Juang and R.G. Harrison Crossflow Microfiltration and Membrane Fouling for Yeast Cell Suspension; S. Redkar and R. Davis Interaction of MBP-β-Galactosidase Fusion Protein with Starch; L. Taladriz and Z. Nikolov Predicting the Solubility of Recombinant Proteins in Escherichia coli; D.L. Wilkinson and R.G. Harrison Evolution of a Phase-Separated, Gravity-Independent Bioractor; P.E. Villeneuve and E.H. Dunlop A Strategy for the Decontamination of Soils Containing Elevated Levels of PCP; S. Ghoshal, S. K. Banelji, and RK. Bajpai Practical Considerations for Implementation of a Field Scale In-Situ Bioremediation Project; J.P. McDonald, CA Baldwin, and L.E. Erickson Parametric Sensitivity Studies of Rhizopus oligosporus Solid Substrate Fermentation; J. Sargantanis, M.N. Karim, and V.G. Murphy, and RP. Tengerdy Production of Acetyl-Xylan Esterase from Aspergillus niger; M.R Samara and J.C. Linden Biological and Latex Particle Partitioning in Aqueous Two-Phase Systems; D.T.L. Hawker, RH. Davis, P.W. Todd, and R Lawson Novel Bioreactor /Separator for Microbial Desulfurization of Coal; H. Gecol, RH. Davis, and J .R Mattoon Effect of Plants and Trees on the Fate, Transport and Biodegradation of Contaminants in the Soil and Ground Water; W. Huang, E. Lee, J.F. Shimp, L.C. Davis, L.E. Erickson, and J.C. Tracy Sound Production by Interfacial Effects in Airlift Reactors; J. Hua, T.-Y. Yiin, LA Glasgow, and L.E. Erickson Soy Yogurt Fermentation of Rapid Hydration Hydrothermal Cooked Soy Milk; P. Tuitemwong, L.E. Erickson, and D.Y.C. Fung Influence of Carbon Source on Pentachlorophenol Degradation by Phanerochaete chrysosportum in Soil; C.-Y.M. Hsieh, RK. Bajpai, and S.K. Banerji Cellular Responses of Insect Cells Spodopiera frugiperda -9 to Hydrodynamic Stresses; P.L.-H. Yeh and RK. Bajpa1 A Mathematical Model for Ripening of Cheddar Cheese; J. Kim, M. Starzak, G.W. Preckshoi, and R.K. Bajpai

(Table 2) Mercury and selenium concentration in the brain stem of wild polar bears (Ursus maritimus), east Greenland

Polar bears (Ursus maritimus) are exposed to high concentrations of mercury because they are apex predators in the Arctic ecosystem. Although mercury is a potent neurotoxic heavy metal, it is not known whether current exposures are of neurotoxicological concern to polar bears. We tested the hypotheses that polar bears accumulate levels of mercury in their brains that exceed the estimated lowest observable adverse effect level (20 µg/g dry wt) for mammalian wildlife and that such exposures are associated with subtle neurological damage, as determined by measuring neurochemical biomarkers previously shown to be disrupted by mercury in other high-trophic wildlife. Brain stem (medulla oblongata) tissues from 82 polar bears subsistence hunted in East Greenland were studied. Despite surprisingly low levels of mercury in the brain stem region (total mercury = 0.36 ± 0.12 µg/g dry wt), a significant negative correlation was measured between N-methyl-D-aspartate (NMDA) receptor levels and both total mercury (r = -0.34, p < 0.01) and methylmercury (r = -0.89, p < 0.05). No relationships were observed among mercury, selenium, and several other neurochemical biomarkers (dopamine-2, gamma-aminobutyric acid type A, muscarinic cholinergic, and nicotinic cholinergic receptors; cholinesterase and monoamine oxidase enzymes). These data show that East Greenland polar bears do not accumulate high levels of mercury in their brain stems. However, decreased levels of NMDA receptors could be one of the most sensitive indicators of mercury's subclinical and early effects.

Stareso MedSeA mesocosm experiment: field and perturbation studies 2012

A joint mesocosm experiment took place in June/July 2012 in Corsica (bay of Calvi, Stareso station;http://www.stareso.com/) as part of the european MedSeA project. Nine mesocosms (52 m**3) were deployed over a 20 days period and 6 different levels of pCO2 and 3 control mesocosms (about 450 µatm), were used, in order to cover the range of pCO2 anticipated for the end of the present century. During this experiment, the potential effects of these perturbations on chemistry, planktonic community composition and dynamics including: eucaryotic and prokaryotic species composition, primary production, nutrient and carbon utilization, calcification, diazotrophic nitrogen fixation, organic matter exudation and composition, micro-layer composition and biogas production were studied by a group of about 25 scientists from 8 institutes and 6 countries. This is one of the first mesocosm experiments conducted in oligotrophic waters. A blog dedicated to this experiment can be viewed at: http://medseastareso2012.wordpress.com/.

Tab. 1: Summary of available data about antifreeze peptides of polar fish

In the blood of Antarctic notothenioid and Arctic gadiform fishes, freezing is inhibited by antifreeze glycopeptide macromolecules (AFGP). These antifreeze molecules are built up of repeating tripeptide units (Ala-Ala-Thr)n, to which the disaccharide fl-D-galactosyl-(1->3)a-N-acetyl-D-galactosamine is linked through the hydroxyl oxygen of the threonyl residue. Species of Liparididae, Zoarcidae, Cottidae and Pleuronectidae synthezise only unglycosylated antifreeze peptides (AFP). It could be demonstrated for the Antarctic silverfish Pleuragramma antarcticum that the synthesis of AFGP is not constitutive but rather regulated by water temperature. Moreover a novel glycopeptid was isolated and characterised from P. antarcticum, the Pleuragramma-antifreeze glycopeptid (PAGP). The level of antifreeze concentration was dependent on the ambient water temperature, the depth of distribution, the life cycle and the evolution of the species. Surprisingly, detectable AFGPs in perciform fish of the Antarctic and gadiform fish of the Arctic and Antarctic could illustrate, that before the continental drift occurred a precursor glycopeptid existed, and that the existence of freezing resistance in some species reflects the past glaciation. The wide distribution and high heterogeneity of AFPs point to the assumption that these peptides are results of cold shock stress responses.

Oligotrophic bacteria from the Antarctic Ocean

From enrichment cultures in dialysis chambers held in natural seawater tanks, 104 strains were isolated and kept in culture. All strains proved to be Gram-negative and psychrotrophic, having optimum growth temperatures of between 20 and 24 °C. Maximal growth temperatures were 30 to 37 °C, or even higher. With 55 isolates, substrate utilizations in Biolog MicroPlates were determined, and the obtained metabolic fingerprints used for clustering. Five groups could be distinguished at the 80% similarity level. Fifteen strains belonged to cluster 1, seven strains to cluster 2, and each of the clusters 3 and 4 contained nine strains. Cluster 5 can be divided into subcluster 5a and 5b, with 6 strains showing a few substrates metabolized, and 9 strains without any reactions, or weak reactions for one or two substrates, respectively. Each cluster could be characterized by specific metabolic fingerprints. Strains from cluster 1 metabolized N-acetyl-D-glucosamine, alpha-hydroxybutyric acid and gamma-hydroxybutyric acid, strains from cluster 2 citric acid, formic acid, thymidine and putrescine, strains from cluster 3 glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-threonine and inosine, whereas strains from cluster 4 metabolized alpha-cyclodextrin and N-acetyl-D-galactosamine, typically. Methylamine was not utilized by the isolates, but strains from cluster 1, 2 and 3 could grow on basal seawater agar. Morphological characteristics and photomicrographs of the oligotrophic strains are presented. Due to their typical morphologies and ampicillin resistence, the nine strains from cluster 3 can be regarded as new species of the genus Planctomyces. These bacteria have not been cultivated before.

Compatibilidad de Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) y Nesidiocoris tenuis (Reuter)(Hemiptera: Miridae), depredadores importantes en cultivos hortícolas protegidos, con nuevas barreras físicas selectivas y modernos plaguicidas

La nueva legislación en materia fitosanitaria se dirige hacia una Gestión Integrada de Plagas (GIP). Estos programas dan preferencia a aquellos métodos más respetuosos y sostenibles con el medio ambiente, siendo piezas claves en ellos el control biológico, el físico y otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos es a veces necesario para el adecuado manejo de plagas en cultivos hortícolas. Por ello, el objetivo general de este estudio es aportar conocimientos para mejorar el control de plagas en cultivos hortícolas, mediante la integración de tres estrategias de lucha: biológica, física y química. Una parte de este trabajo ha consistido en el estudio de los posibles efectos que mallas tratadas con insecticida (bifentrin) pudieran provocar mediante diferentes ensayos de laboratorio, invernadero y campo, en los enemigos naturales Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) (depredador de trips), Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) (depredador de mosca blanca y Tuta absoluta (Meirick) (Lepidoptera: Gelechiidae)), y otros agentes de biocontrol comúnmente usados en cultivos hortícolas protegidos. Este tipo de mallas se han empleado con éxito en entomología médica para controlar mosquitos vectores de la malaria, y actualmente se está trabajando en su desarrollo para uso agrícola como método de exclusión, y método directo de control de plagas. En los ensayos realizados en laboratorio, O. laevigatus y N. tenuis no fueron capaces de detectar la presencia de bifentrin en el ensayo de preferencia. Además, no se produjo mortalidad a corto plazo (72 horas) en ambos chinches depredadores. Por el contrario, se registró una elevada mortalidad cuando se expusieron por contacto a la malla tratada durante 72 horas en cajas de dimensiones reducidas (10 cm de diámetro X 3 cm de altura). En ensayos llevados a cabo bajo condiciones más reales de exposición, en un invernadero experimental con jaulas de 25 X 25 X 60 cm de altura, no se produjo ningún efecto en la mortalidad a corto plazo (72 horas) o en los parámetros reproductivos de O. laevigatus y N. tenuis. Finalmente, en ensayos de campo realizados en túneles semi-comerciales (8 m de largo X 6,5 m de ancho X 2,6 m de altura), ni las condiciones ambientales [temperatura, humedad relativa, radiación ultravioleta (UV) y fotosintéticamente activa (PAR)], ni los enemigos naturales, se vieron afectados por la presencia de la malla tratada con bifentrin en el cultivo. Sin embargo, los resultados no fueron concluyentes, debido al bajo establecimiento de los agentes de biocontrol liberados. Por lo tanto, más estudios son necesarios en invernaderos comerciales para confirmar los resultados preliminares de compatibilidad. Además, en este trabajo se han evaluado los efectos letales (mortalidad) y subletales (parámetros reproductivos) de seis modernos insecticidas sobre los chinches depredadores O. laevigatus y N. tenuis, mediante ensayos de laboratorio y persistencia. Los ensayos se realizaron por contacto residual, aplicando los insecticidas a la dosis máxima de campo sobre placas de cristal (laboratorio) o plantas (persistencia). Los productos fitosanitarios se seleccionaron por representar a un grupo de modernos plaguicidas con modos de acción en principio más selectivos para los enemigos naturales que antiguos plaguicidas como organoclorados, oroganofosforados o carbamatos, y por su uso frecuente en cultivos hortícolas donde O. laevigatus y N. tenuis están presentes. Todos ellos están incluidos o en proceso de inclusión en la lista comunitaria de sustancias activas para uso agrícola, Anexo I de la Directiva 91/414/CEE: abamectina y emamectina (avermectinas neurotóxicas, activadoras del canal del cloro), deltametrina (piretroide neurotóxico, modulador del canal del sodio, control positivo), flubendiamida (neurotóxico, modulador del receptor de rianodina), spinosad (naturalito neurotóxico, agonistas/antagonistas del receptor de nicotínico acetilcolina) y spiromesifen (inhibidor de la acetil CoA carboxilasa). El estudio mostró que O. laevigatus fue más susceptible a los insecticidas que N. tenuis. Además, los resultados revelaron que flubendiamida y spiromesifen fueron compatibles con los dos enemigos naturales estudiados, y por tanto se podrían usar en programas de GIP. Por el contrario, los insecticidas abamectina, deltametrina, emamectina y spinosad no fueron selectivos para ninguno de los chinches depredadores. Sin embargo, los estudios de persistencia demostraron que a pesar de que estos insecticidas no proporcionaron selectividad fisiológica, pueden proporcionar selectividad ecológica en algunos casos. Abamectina, deltametrina, emamectina y spinosad podrían ser compatibles con N. tenuis si el enemigo natural es introducido en el cultivo 4 días después de su aplicación. En el caso de O. laevigatus, abamectina, deltametrina y spinosad se clasificaron como persistentes, por lo tanto es necesario completar el estudio con experimentos de semi-campo y campo que determinen si es posible su uso conjunto en programas de GIP. Por otro lado, emamectina podría ser compatible con O. laevigatus si el enemigo natural es introducido en el cultivo 7 días después de su aplicación. Por último, se ha comprobado la selectividad de tres insecticidas aceleradores de la muda (MACs) (metoxifenocida, tebufenocida y RH-5849) sobre O. laevigatus y N. tenuis. Además de realizar estudios para evaluar la toxicidad en laboratorio de los insecticidas por contacto residual e ingestión (principal modo de acción de los MAC´s), se extrajo RNA de los insectos y con el cDNA obtenido se secuenció y clonó el dominio de unión al ligando (LBD) del receptor de ecdisona correspondiente a O. laevigatus (OlEcR-LBD) y N. tenuis (NtEcR-LBD). Posteriormente, se obtuvo la configuración en tres dimensiones del LBD y se estudió el acoplamiento de las moléculas de los tres insecticidas en la cavidad que forman las 12 α-hélices que constituyen el EcR-LBD. En el caso de N. tenuis se debe mencionar que no fue posible la obtención de la secuencia completa del LBD. Sin embargo, se obtuvo una secuencia parcial (hélice 6-hélice 11), que mostró una alta conservación de aminoácidos con respecto a la obtenida en O. laevigatus. Los ensayos de toxicidad mostraron que metoxifenocida, tebufenocida y RH-5849 no produjeron ningún efecto nocivo en ambos depredadores. Además, los estudios de modelado por homología y acoplamiento molecular llevados a cabo con O. laevigatus, también indicaron que los MACs no produjeron ningún efecto deletéreo en este enemigo natural. Por lo tanto, estos compuestos pueden ser aplicados de manera segura en programas de GIP en los cuales O. laevigatus y N. tenuis estén presentes. ABSTRACT The new pesticide legislation on pest control is aimed at integrated pest management (IPM). These programs are based on the most environmentally sustainable approaches, where biological, physical control and other non-chemical methods are the cornerstone. However, selective pesticides are often required for pest management on horticultural crops. Therefore, the main goal of this study is to provide knowledge to improve pest control on horticultural crops through the integration of three strategies: biological, physical and chemical. Firstly, the effects of insecticide treated nets (bifenthrin) were evaluated in different laboratory, greenhouse and field experiments on the natural enemies Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) (predator of thrips), Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) (predator of whiteflies and Tuta absoluta (Meirick) (Lepidoptera: Gelechiidae)), and other biocontrol agents commonly used on protected horticultural crops. These types of nets have been successfully used in medical entomology to control mosquito malaria vectors, and work is currently being done on their use as exclusion barriers and as a direct method of pest control in agriculture. In experiments made under laboratory conditions, O. laevigatus and N. tenuis were not able to detect the presence of bifenthrin in a dual-choice test. Furthermore, no shortterm mortality (72 hours) was recorded on both predatory bugs. In contrast, a high mortality rate was found when they were exposed by contact to the bifenthrin-treated net for 72 hours in small cages (10 cm diameter X 3 cm high). In assays carried out under more realistic conditions of exposure, in an experimental greenhouse with cages of 25 X 25 X 60 cm high, short-term mortality (72 hours) and reproductive parameters were not affected. Lastly, in field experiments carried out in semi-commercial tunnels (8 m long X 6.5 m width X 2.6 m high), neither environmental conditions [temperature, relative humidity, ultraviolet (UV) and photosynthetically active radiation (PAR)] nor natural enemies were affected by the presence of the bifenthrin-treated net on the crop. However, results were not conclusive, mainly due to a low settlement of the released biocontrol agents, and further studies are needed in commercial greenhouses to confirm our preliminary results of compatibility. Secondly, the lethal (mortality) and sublethal effects (reproductive parameters) of six modern pesticides on the predatory bugs O. laevigatus and N. tenuis has been evaluated through laboratory and persistence experiments. Trials were carried out by residual contact, applying the insecticides to the maximum field recommended concentration on glass plates (laboratory) or plants (persistence). Insecticides were chosen as representatives of modern pesticides with a more selective mode of action on natural enemies than organochlorine, organophosphorus and carbamate insecticides. Moreover, they were also chosen because of their frequent use on horticultural crops where O. laevigatus and N. tenuis are present. All of them have been included or have been requested for inclusion in the community list of active substances on the agricultural market, Annex I of the European Directive 91/414/EEC: abamectin and emamectin (neurotoxic avermectins, chloride channel activators), deltamethrin (neutotoxic pyrethroid, sodium channel modulator, positive commercial standard), flubendiamide (neurotoxic, rianodine receptor modulator), spinosad (neurotoxic naturalyte, nicotinic acetylcholine receptor allosteric activator) and spiromesifen (inhibitors of acetyl CoA carboxylase). The study showed that O. laevigatus was more susceptible to all the studied pesticides than N. tenuis. In addition, the research results indicated no impact of flubendiamide and spiromesifen on the two natural enemies studied under laboratory conditions. Consequently, both pesticides are candidates to be included in IPM programmes where these biocontrol agents are present. On the other hand, abamectin, deltamethrin, emamectin and spinosad were not selective for both predatory bugs in laboratory experiments. However, persistence test demonstrated that in spite of the lack of physiological selectivity, these pesticides can provide ecological selectivity in some cases. Abamectin, deltamethrin, emamectin and spinosad could be compatible with N. tenuis if the mirid bug is released 4 days after the insecticide treatment on the crop. With regard to O. laevigatus, abamectin, deltamethrin and spinosad were classified as persistent in our assays, thus the study should be completed with semi-field and field experiments in order to ascertain their possible joint use in IPM programs. In contrast, emamectin could be compatible with O. laevigatus if the pirate bug is released 7 days after the insecticide treatment on the crop. Finally, the selectivity of three moulting accelerating compounds (MACs) (methoxyfenozide, tebufenozide and RH-5849) has also been evaluated on O. laevigatus and N. tenuis. In addition to laboratory experiments to evaluate the toxicity of the insecticides by residual contact and ingestion, molecular approaches were used as well. RNA of both insects was isolated, cDNA was subsequently synthesized and the complete sequence of the ligand binding domain (LBD) of the ecdysone receptor of O. laevigatus (OlEcR-LBD) and N. tenuis (NtEcR-LBD) were determined. Afterwards, the three dimensional structure of LBD was constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the EcRLBD was performed. In the case of N. tenuis, it should be noted that in spite of intensive efforts, we did not manage to complete the sequence for the LBD.However, a partial sequence of the LBD was obtained (helix 6-helix 11), and a strong conservation between the amino acids of N. tenuis and O. laevigatus was observed. Results showed no biological activity of methoxyfenozide, tebufenozide and RH-5849, on both predatory bugs. Moreover, modeling of the OlEcR-LBD and docking experiments also suggested that MACs were devoid of any deleterious effect on O. laevigatus. Therefore, our results indicate that these compounds could be safely applied in IPM programs in which O. laevigatus and N. tenuis are present.