Effects of dibutyl phthalate and di‐ethylhexyl phthalate on acetylcholinesterase activity in bagrid catfish,Pseudobagrus fulvidraco (Richardson)

Acetylcholinesterase (AChE) activity is a well‐known biomarker for exposure to organophosphate or carbamate compounds in aquatic organisms. However, the effect of dibutyl phthalate (DBP) and di‐ethylhexyl phthalate (DEHP), widely used as a plasticizer, on the change of AChE activity is not yet known. Bagrid catfish Pseudobagrus fulvidraco were administrated with 100, 500 and 1000 mg kg^?1 diet of DBP or DEHP and the effects on AChE activity were assessed in the liver, gill, kidney, heart, brain, muscle and eye of the exposed fish. All tissues contained different background AChE activity in non‐treated bagrid catfish: the highest was observed in the brain, followed by muscle, heart, and kidney. The enzyme activities in various tissues were significantly inhibited after exposure to DBP or DEHP in a concentration‐dependent manner, especially in brain and muscle. A similar, but less pronounced, inhibition was also observed in liver and kidney when exposed to DBP and DEHP. Although AChE activity in gill and heart was also affected by DBP and DEHP, the decrease in these organs was least marked in these organs. Exposure to 1000 mg kg^?1 led to mortalities of 8.0% with DBH and 14% with DEHP; both seemed to be ascribable to phthalate toxicity. This study is the first report that the measurement of AChE activity in bagrid catfish is a valuable biomarker of DBP and DEHP exposure. This biomarker could be incorporated into a battery of biomarkers to strengthen the confidence with which ecotoxicologists can assess the impact of phthalate ester pollution in the aquatic environment.     

Di‐2‐ethylhexyl phthalate (DEHP) exposure disturbs lipid metabolism in juvenile yellow catfish Tachysurus fulvidraco

This study was conducted to determine the mechanism by which di‐2‐ethylhexyl phthalate (DEHP) exposure influences lipid metabolism of juvenile yellow catfish Tachysurus fulvidraco. Fish were exposed to three DEHP concentrations (0, 0·1 and 0·5 mg l^?1 DEHP) for 8 weeks. Fatty acid synthase (FAS) activity significantly decreased with increasing DEHP concentrations, the highest value was in the Tween control group, whereas the lowest activities of carnitine palmitoyltransferase (CPT) and lipoprotein lipase (LPL) were in this group. The messenger (m)RNA levels of 6‐phospho‐gluconate dehydrogenase (6PGD), FAS and acetyl‐CoA carboxylase a (ACCa) significantly increased with increasing DEHP concentration, the highest values were in the 0·5 mg l^?1 DEHP group. The mRNA level of peroxisome proliferator‐activated receptor γ (PPARγ) was lower in Tween control than in fish exposed to 0·1 and 0·5 mg l^?1 DEHP. The highest mRNA level of ACCb was in the 0·1 mg l^?1 DEHP group. These results indicate that DEHP exposure can disturb lipid metabolism at the enzymatic and mRNA levels in Pelteobagrus fulvidraco.     

Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin‐, K+‐ and Fe2+‐dependent key enzyme of anaerobic phthalate degradation

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2‐dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report on the first purification and characterization of a phthaloyl‐CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD‐family of (de)carboxylases and contains prenylated FMN (prFMN), K⁺ and, unlike other UbiD‐like enzymes, Fe²⁺ as cofactors. The latter is suggested to be involved in oxygen‐independent electron‐transfer during oxidative prFMN maturation. Either oxidation to the Fe³⁺‐state in air or removal of K⁺ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe²⁺/K⁺‐complex in PCD. The PCD‐catalysed reaction was essentially irreversible: neither carboxylation of benzoyl‐CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl‐CoA with ¹³C‐bicarbonate was observed. PCD differs in many aspects from prFMN‐containing UbiD‐like decarboxylases and serves as a biochemically accessible model for the large number of UbiD‐like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.     

Spectroscopic and molecular simulation studies on the interaction of di‐(2‐ethylhexyl) phthalate and human serum albumin

Di‐(2‐ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in industrial production, but may have a potential health risk. In this study, the binding characteristics of DEHP with human serum albumin (HSA) in aqueous solution at pH 7.4 were determined using UV/vis absorption, fluorescence, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD), along with a molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by DEHP was static. The calculated thermodynamic parameters indicated that hydrophobic forces played a predominant role in formation of the DEHP–HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments and denaturation studies showed that the binding of DEHP to HSA primarily took place in subdomain IIA of HSA, and molecular docking results further corroborated the binding sites. The synchronous fluorescence, UV/vis absorption, FTIR and CD spectra revealed that the addition of DEHP induced changes in the secondary structure of HSA. Protein surface hydrophobicity (PSH) tests indicated that DEHP binding to HSA caused an increase in the PSH. Moreover, the effects of some metal ions on the binding constant of DEHP − HSA interaction were also investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase

The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.     

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl‐CoA intermediate during anaerobic phthalate degradation

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen‐independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from ‘Aromatoleum aromaticum’: a highly specific succinyl‐CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl‐CoA formed by SPT accumulated only to sub‐micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half‐life of around 7 min. Upon addition of excess phthaloyl‐CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl‐CoA formation. In conclusion, a massive overproduction of PCD in phthalate‐grown cells to concentrations >140 μM was observed that allowed for efficient phthaloyl‐CoA conversion at concentrations 250‐fold below the apparent K_m‐value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.     

Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia

An enzymatic system has been isolated that catalyzes dihydroxylation of phthalate to form 1,2-dihydroxy-4,5-dicarboxy-3,5-cyclohexadiene with consumption of NADH and O2. This system is comprised of two proteins: a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity and a nonheme iron protein with oxygenase activity. Phthalate oxygenase is a large (approximately 217 kDa) protein composed of apparently identical 48-kDa monomers. The active enzyme has one Rieske-type [2Fe-2S] center and one mononuclear iron/monomer. Removal of the mononuclear iron by incubation with EDTA or with o-phenanthroline inhibits oxygenation; ferrous ion completely restores activity. No other metals are effective. Phthalate oxygenase is specific for phthalate or other closely related compounds. However, only phthalate is tightly coupled to NADH oxidation and O2 consumption with a stoichiometry of 1:1:1. Phthalate oxygenase is chemically competent to oxygenate phthalate when artificially supplied with reducing equivalents and O2. Phthalate oxygenase reductase is required, however, for efficient catalytic activity. The reductase is a monomeric 34-kDa flavo-iron-sulfur protein containing FMN and a plant-ferredoxin-type [2Fe-2S] center in a 1:1 ratio. Phthalate oxygenase reductase is specific for NADH but can pass electrons to a variety of acceptors, including: phthalate oxygenase, cytochrome c, ferricyanide, and dichlorophenolindophenol. This system is similar to other bacterial oxygenase systems involved in aromatic degradation including: benzoate dioxygenase, toluene dioxygenase, benzene dioxygenase, and 4-methoxybenzoate demethoxylase. However, phthalate oxygenase can be isolated in large quantities and is more stable than most other such systems.     

Denitrifying degradation of dimethyl phthalate

Results of batch experiments on the denitrifying degradation of dimethyl phthalate (DMP) was most favorable at pH 7–9 and 30–35°C. DMP was first degraded to monomethyl phthalate (MMP), which was in turn degraded to phthalate before complete mineralization. There was no fatty acid residue in the mixed liquor throughout the experiments. The maximum specific degradation rates were 0.32 mM/(gVSS·h) for DMP, 0.19 mM/(gVSS·h) for MMP, and 0.14 mM/(gVSS·h) for phthalate. About 86% of available electron in DMP was utilized for denitrification; the remaining 14% was presumable conserved in the new biomass with an estimated yield of 0.17 mg/mg DMP. Based on 16S rDNA analysis, the denitrifying sludge was mainly composed of β-subdivision and α-subdivision of Proteobacteria (33 and 5 clones out of a total of 43 clones, respectively), plus some Acidobacteria. Using a primer set specifically designed to amplify the denitrification nirK gene, 10 operational taxonomy units (OTUs) were recovered from the clone library. They clustered into a group in the α-subdivision of Proteobacteria most closely related to denitrifier Bradyrhizobium japonicum USDA110 and several environmental clones.     

Synthesis of enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate and endo‐2‐norbornyl‐N‐n‐butylcarbamate for stereoselective inhibition of acetylcholinesterase

The acetylcholinesterase inhibition by enantiomers of exo‐ and endo‐2‐norbornyl‐N‐n‐butylcarbamates shows high stereoselelectivity. For the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐exo‐2‐norbornyl‐N‐n‐butylcarbamates, the R‐enantiomer is more potent than the S‐enantiomer. But, for the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates, the S‐enantiomer is more potent than the R‐enantiomer. Optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates are synthesized from condensations of optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norborneols with n‐butyl isocyanate, respectively. Optically pure norborneols are obtained from kinetic resolutions of their racemic esters by lipase catalysis in organic solvent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

The crystal structure of Z‐Gly‐Aib‐Gly‐Aib‐OtBu

The synthetic peptide Z‐Gly‐Aib‐Gly‐Aib‐OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petroleum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Structural Database. The first Gly residue assumes a semi‐extended conformation (φ ±62°, ψ ?131°). The right‐handed peptide folds in two consecutive β‐turns of type II' and type I or an incipient 3₁₀‐helix, and the left‐handed counterpart folds accordingly in the opposite configuration. In the crystal lattice, one molecule is linked to four neighbors in the ab‐plane via hydrogen bonds. These bonds form a continuous network of left‐ and right‐handed molecules. The successive ab‐planes stack via apolar contacts in the c‐direction. An ethyl acetate molecule is situated on and close to the fourfold axis. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Degradation of diphenyl phthalate by <Emphasis Type="Italic">Sphingomonas chungbukensis</Emphasis>

More than 80% of diphenyl phthalate (DPP) at 100 mg l⁻¹ was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30°C within 48 h. The maximum specific degradation rate was 5 mg DPP l⁻¹ h⁻¹. It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.     

Homo‐C‐nucleoside analogs IV. Synthesis of 4‐(2,5‐anhydro‐D‐altro‐pentitol‐1‐yl)‐2‐phenyl‐2H‐1,2,3‐triazole homo‐C‐nucleoside. CD assignment of the absolute configuration of the carbon bridge

Dehydrative cyclization of 4‐(D‐altro ‐pentitol‐1‐yl)2‐phenyl‐2H ‐1,2,3‐triazole in basic medium with one moler equivalent of p‐toluene sulfonyl chloride in pyridine solution gave the homo‐C‐ nucleoside 4‐(2,5‐anhydro‐D‐altro ‐1‐yl)‐2‐phenyl‐2H ‐1,2,3‐triazole. The structure and anomeric configuration was determined by acylation, nuclear magnetic resonance (NMR), and mass spectroscopy. The stereochemistry at the carbon bridge of homo‐C‐ nucleoside 2‐phenyl‐2H ‐1,2,3‐triazoles was determined by circular dichroism (CD) spectroscopy.     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Crystal structure of D‐glycero‐Β‐D‐manno‐heptose‐1‐phosphate adenylyltransferase from Burkholderia pseudomallei

The crystal structure of HldC from B. pseudomallei (BpHldC), the fourth enzyme of the heptose biosynthesis pathway, has been determined. BpHldC converts ATP and d ‐glycero‐β‐d ‐manno‐heptose‐1‐phosphate into ADP‐d ‐glycero‐β‐d ‐manno‐heptose and pyrophosphate. The crystal structure of BpHldC belongs to the nucleotidyltransferase α/β phosphodiesterase superfamily sharing a common Rossmann‐like α/β fold with a conserved T/HXGH sequence motif. The invariant catalytic key residues of BpHldC indicate that the core catalytic mechanism of BpHldC may be similar to that of other closest homologues. Intriguingly, a reorientation of the C‐terminal helix seems to guide open and close states of the active site for the catalytic reaction.     

Some Aspects of Continuity and Change Among Anthropologists in Australia or‘He‐Who‐Eats‐From‐One‐Dish‐With‐Us‐With‐One‐Spoon’

Since the future of anthropology in Australia is clouded, the address takes a look at where it has been coming from. Rather than a distinctive regional school, the discipline in Australia has been part of anthropology in the UK and the USA. In common with anthropology elsewhere, it lacks a distinctive theoretical stance, but draws on the theory current in the other social sciences. Recognising that what makes anthropology ‘special’ is the field work experience, the address reflects on the history and nature of this practice.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.