Benzophenone-3 does not Cause Oxidative Stress or B-esterase Inhibition During Embryo Development of Octopus maya (Voss and Solís Ramírez, 1966).

Benzophenone-3 (BP-3) is an active ingredient in sunscreen lotions and personal-care products that protects against the damaging effects of ultraviolet rays. Given its worldwide dissemination, it has been linked with harmful effects on aquatic biota; however, its impact is not fully understood calling for further studies. To understand the impacts on an important economically and ecologically species, we evaluated the toxicity of BP-3 during the embryonic development of Octopus maya. Embryos were exposed to increasing concentrations of up to 500 µg BP-3/L until hatching. Antioxidant enzyme activities, oxidative-stress indicators, and B-esterases activities were measured at different developmental phases (organogenesis, activation, and growth). There were no significant differences between treatments, suggesting the lack of production of toxic metabolites that may be related to a protective chorion, an underdeveloped detoxification system, and the experimental conditions that limited phototoxicity.

Effect of microplastics on the activity of carboxylesterase and phosphatase enzymes in Scinax squalirostris tadpoles.

Microplastics (MPs) are critical emerging pollutants around the world. There is a growing interest in the effects of MP ingestion, non-digestion, and toxicity on aquatic organisms. Amphibian tadpoles are the vertebrate group that has received the least attention regarding this issue. The aim of the present study was to determine the ingestion of polyethylene MPs by Scinax squalirostris tadpoles by atomic force microscopy (AFM) and to evaluate the activities of carboxylesterase (CbE, using 4-naphthyl butyrate-NB-, and 1-naphthyl acetate -NA- as substrates) and alkaline phosphatase (ALP) under MP exposure. Enzyme activities were analyzed spectrophotometrically at 2 and 10 days of exposure. Tadpoles were exposed to two different treatments during 10 days: a negative control (CO, dechlorinated water) and MP (60 mg L-1). AFM images of the digestive contents of tadpoles revealed the presence of MPs. After 10 days of MP exposure, CbE (NB) activity was significantly higher and CbE (NA) activity was significantly lower in MP treatments than in controls. ALP activity decreased in MP treatments after 2 and 10 days of exposure. The detection of MP particles in the intestinal contents and the effects on metabolic enzymes in a common frog species evidenced the potential health risk of MP to aquatic vertebrates. Thus, the differential response in enzymes and substrates demonstrate the need for considering the complex effects of contaminants and nutrients on ecosystems for ecotoxicological risk characterization.

A Fungus-Inducible Pepper Carboxylesterase Exhibits Antifungal Activity by Decomposing the Outer Layer of Fungal Cell Walls.

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants.

MAIN CONCLUSION: Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling.

GLIP1 was isolated previously from Arabidopsis, as a salicylic acid-responsive secreted GDSL lipase that functions in resistance to Alternaria brassicicola [I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17 (2005) 2832-2847.]. To extend our knowledge of the roles played by GLIPs in Arabidopsis, we conducted functional studies of another family member, GLIP2. GLIP2 transcripts were expressed in young seedlings, as well as in the root and stem tissues of mature plants. GLIP2 transcript levels were elevated by treatment with salicylic acid, jasmonic acid and ethylene. Recombinant GLIP2 proteins possessed lipase and anti-microbial activities, inhibiting germination of fungal spores. In comparison to wild type plants, T-DNA insertion glip2 mutants exhibited enhanced auxin responses, including increased lateral root formation and elevated AUX/IAA gene expression. When challenged with the necrotropic bacteria Erwinia carotovora, glip2 mutants exhibited more susceptible phenotypes than wild type plants. These results suggest that GLIP2 plays a role in resistance to Erwinia carotovora via negative regulation of auxin signaling.

Metabolism and autoradiographic evaluation of [(18)F]FE@CIT: a Comparison with [(123)I]beta-CIT and [(123)I]FP-CIT.

PURPOSE: Since the late 1980s, cocaine analogues based on the phenyltropane structure, such as [(11)C]CFT and [(123)I]beta-CIT have been used for the imaging of the dopamine transporter. FE@CIT (fluoropropyl ester) and FP-CIT (N-fluoropropyl derivative) are further analogues. The aim of this study was to (1) evaluate and compare the metabolic stability of beta-CIT, FP-CIT and FE@CIT against carboxyl esterases and (2) evaluate selectivity of [(18)F]FE@CIT compared to [(123)I]beta-CIT and [(123)I]FP-CIT using autoradiography. METHODS: In vitro enzymatic hydrolysis assays were performed using different concentrations of beta-CIT, FE@CIT and FP-CIT with constant concentrations of carboxyl esterase. Autoradiography was performed on coronal 20-microm rat brain sections incubated with different radioactivity concentrations of [(123)I]beta-CIT, [(123)I]FP-CIT or [(18)F]FE@CIT and, additionally, with 3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile [serotonin transporter (SERT)] and nisoxetine [norepinephrine transporter (NET)] for blocking experiments. RESULTS: In vitro assays showed Michaelis-Menten constants of 175 micromol (beta-CIT), 183 micromol (FE@CIT) and 521 micromol (FP-CIT). Limiting velocities were 0.1005 micromol/min (beta-CIT), 0.1418 micromol/min (FE@CIT) and 0.1308 micromol/min (FP-CIT). This indicates a significantly increased stability of FP-CIT, whereas carboxyl esterase stability of beta-CIT and FE@CIT showed no significant difference. Autoradiographic analyses revealed a good correlation between dopamine transporter (DAT)-rich regions and the uptake pattern of FE@CIT. Blocking experiments showed a higher DAT selectivity for [(18)F]FE@CIT than for the other two tracers. CONCLUSION: We found that (1) the metabolic stability of FE@CIT was comparable to that of beta-CIT, whereas FP-CIT showed higher resistance to enzymatic hydrolysis; and (2) the overall uptake pattern of [(18)F]FE@CIT on brain slices was comparable to that of [(123)I]beta-CIT and [(123)I]FPCIT. After blocking of NET and SERT binding, a significantly higher DAT selectivity was observed for [(18)F]FE@CIT. Hence, [(18)F]FE@CIT may be of interest for further clinical application.

Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3.

Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [(3)H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters approximately 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway.

Detoxification of organophosphorus compounds by recombinant carboxylesterase from an insecticide-resistant mosquito and oxime-induced amplification of enzyme activity.

Currently, bioremediation is a promising approach to the degradation of environmental pollutants. Here we describe the application of the recombinant insecticide-resistant mosquito carboxylesterase B1 to detoxify organophosphorous compounds. However, this approach has a major limitation: 1:1 stoichiometry of the enzyme detoxification of those organophosphorous compounds containing no carboxyl ester bonds, such as paraoxon, chlorpyrifos etc. To improve the effectiveness of the enzymatic detoxification of organophosphorous compounds, we used a combination of carboxylesterase B1 with the uncharged oxime diacetylmonoxime. It was demonstrated that the repeated addition of 20 times the molar concentration of paraoxon to carboxylesterase B1 every 2 h in the presence of 4 mM diacetylmonoxime did not result in significant inhibition of the enzyme. The stoichiometry of enzyme detoxification was higher than 45:1 and 20:1 for paraoxon and chlorpyrifos, respectively. The kinetic experiments on reactivation of organophosphorus compound-inhibited carboxylesterase B1 showed that the half-life for paraoxon- and chlorpyrifos-inhibited carboxylesterase reactivation is 0.75 and 0.88 h, respectively. Using the recombinant insecticide-resistant mosquito carboxylesterase with oxime is an effective approach for detoxification of organophosphorous compounds.

The enzymology of sludge solubilisation utilising sulphate reducing systems: the role of lipases.

The first stage in the degradation and recycling of particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the sulphidogenic environment. An investigation into the enzymology of these processes has shown that lipase enzyme activities were found predominantly associated with the organic particulate matter of the sewage sludge. Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant. pH and temperature optimisation studies showed optima at between 6.5 and 8 and 50-60 degrees C, respectively. All the lipase enzymes from the methanogenic bioreactors indicated extensive stability for at least an hour at their respective optimum temperatures and pH; sulphidogenic lipases reflected limited stability at these temperatures and pH during this time period. Though sulphate showed inhibitory properties towards lipases both sulphide and sulphite appeared to enhance the activity of the enzymes. It is argued that these sulphur species, liberated at different times during the sulphate reduction process, disrupt the integrity of the organic particulate floc by neutralising acidic components on the surface. The release of further entrapped enzymes from the organic particulate matter results in a subsequent enhancement of hydrolysis of polymeric material.