Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.     

Evaluation of halophilic microalgae isolated from Rabigh Red Sea coastal area for biodiesel production: Screening and biochemical studies

In the present study, different water samples from Red Sea coastal area at Rabigh city, Saudi Arabia were studied for their dominant algal species. Microalgal isolation was carried out based on dilution method and morphologically examined using F/2 as a growth medium. Dry weight and main biochemical composition (protein, carbohydrates, lipids) of all species were performed at the end of the growth, and biodiesel characteristics were estimated. Nannochloropsis sp., Dunaliella sp., Tetraselmis sp., Prorocentrum sp., Chlorella sp., Nitzschia sp., Coscinodiscus sp., and Navicula sp. were the most dominant species in the collected water samples and were used for further evaluation. Nannochloropsis sp. surpassed all other isolates in concern of biomass production with the maximum recorded dry weight of 0.89 g L^?1, followed by Dunaliella sp. (0.69 g L^?1). The highest crude protein content was observed in Nitzschia sp. (38.21%) and Dunaliella sp. (18.01%), while Nannochloropsis sp. showed 13.38%, with the lowest recorded lipid content in Coscinodiscus sp. (10.09%). Based on the growth, lipid content, and biodiesel characteristics, the present study suggested Dunaliella sp. and Nitzschia sp. as promising candidates for further large-scale biodiesel production.     

Ontogeny of the jaw and maxillary barbel musculature in the armoured catfish families Loricariidae and Callichthyidae (Loricarioidea,Siluriformes), with a discussion on muscle homologies

The neotropical loricarioid catfishes include six families, the most species‐rich of which are the Callichthyidae and the Loricariidae. Loricariidae (suckermouth armoured catfishes) have a highly specialized head morphology, including an exceptionally large number of muscles derived from the adductor mandibulae complex and the adductor arcus palatini. Terminology of these muscles varies among the literature, and no data exist on their ontogenetic origin. A detailed examination of the ontogeny of both a callichthyid and a loricariid representative now reveals the identity of the jaw and maxillary barbel musculature, and supports new hypotheses concerning homologies. The adductor mandibulae muscle itself is homologous to the A1‐OST and A3′ of basal catfishes, and the A3′ has given rise to the newly evolved loricariid retractor veli as well. The A2 and A3″ have resulted in the retractor tentaculi of Callichthyidae and the retractor premaxillae of Loricariidae. Thus, these two muscles are shown to be homologous. In Loricariidae, the extensor tentaculi consists of two separate muscles inserting on the autopalatine, and evidence is given on the evolutionary origin of the loricariid levator tentaculi (previously and erroneously known as retractor tentaculi) from the extensor tentaculi, and not the adductor mandibulae complex. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 76–96.     

褐菖鲉肝CYP 1A作为生物标志物监测厦门海域石油污染状况

以褐菖鲉为实验鱼类,以鱼肝微粒体CYP1A 生物标志物(EROD活性和CYP1A蛋白表达量)为指标,在厦门海域开展了两次野外监测实验,研究EROD活性和CYP1A蛋白表达量的变化,以及它们与海水和沉积物中石油类和重金属含量之间的相关性。结果表明,在现场属于一类海水的石油类浓度(0.0121-0.0242 mg/L)条件下,石油类就能够显著诱导褐菖鲉肝EROD活性和CYP1A蛋白表达量,鱼肝EROD活性和CYP1A蛋白表达量与海水中石油类含量均呈现极显著正相关,CYP1A蛋白表达量比EROD活性较为敏感和稳定。此外,在监测实验中,尚未发现这两种生物标志物受所监测海区的海水和沉积物重金属含量的影响。因此,利用褐菖鲉肝微粒体EROD活性和CYP1A蛋白表达量作为生物标志物监测海洋石油类及其PAHs污染是可行的,在海洋环境石油类污染监测及其生化效应评价中具有重要的应用价值。而且,把这两种生物标志物结合起来加以研究并推广应用将更有意义。     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR^null, POR^wt, POR^YH, and POR^VE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR^null, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR^YH and POR^VE models than in POR^wt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.     

Modern Distribution of Armored Catfishes (Siluriformes: Loricariidae) in Central Vietnam

The modern distribution of South American armored catfishes (Loricariidae) is studied in three provinces in Central Vietnam. In the investigated area, representatives of the genus Pterygoplichthys occur, and at present they have moved into all main river basins. During the last 2–5 years, alien species have entered into many water bodies isolated from the places of their first discovery by natural and manmade barriers. This indicates the important role of the human factor for the expansion of loricariids in the region.     

Complexes of Trypanosoma cruzi Sterol 14α-Demethylase (CYP51) with Two Pyridine-based Drug Candidates for Chagas Disease: STRUCTURAL BASIS FOR PATHOGEN SELECTIVITY*

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.     

New and little known species of the genus Zorochros Thomson 1859 (Coleoptera: Elateridae) from Palaearctic and Oriental Region

Sixteen new species of the genus Zorochros Thomson 1859 from China, Indonesia, Malaysia, Nepal and Thailand are described and illustrated, a key to the species of the Zorochros indicus group is given, and new records on already known species are provided. New species: Z. ahrensi n. sp., Z bingkorensis n. sp., Z. dabieshanensis n. sp., Z. dolini n. sp., Z. hartmanni n. sp., Z. hubeiensis n. sp., Z. karnaliensis n. sp., Z. magnificus n. sp., Z. naniensis n. sp., Z. nigredos n. sp., Z. platiai n. sp., Z schawalleri n. sp., Z. schmidti n. sp., Z. senaroensis n. sp., Z. theodori n. sp., and Z. tongshanensis n. sp.     

A revision of the genus Paronthobium from New Caledonia (Coleoptera: Scarabaeidae: Scarabaeinae: Epilissini)

A revision of the New Caledonian genus Paronthobium Paulian 1984 is presented. Anonthobium Paulian 1984 is synonymized with Paronthobium Paulian 1984 after evaluating the diagnostic characters originally introduced by Paulian to separate them. Upon examination of the type specimens, Onthobium caledonicum Paulian 1935 is separated from O. simplex Fauvel 1903 and restored as valid. Anonthobium moui Paulian 1984 is synonymized with Anonthobium micros Paulian 1984. Fifteen new species are described: P. adio n. sp., P. daghfousi n. sp., P. dierkensi n. sp., P. farino n. sp., P. iac n. sp., P. julieni n. sp., P. memaoya n. sp., P. minutum n. sp., P. nasutum n. sp., P. orientale n. sp., P. peckorum n. sp., P. petchecara n. sp., P. subdentatum n. sp., P. taom n. sp. and P. tchingou n. sp. The genus Paronthobium is now composed of 22 species. Illustrations of parameres and of male protibiae are provided for each species. Distribution maps and a key to species are given.     

Catalytic properties of CYP1A isoforms in the liver of an agnathan (Lampetra fluviatilis) and two species of teleost (Pleuronectes flesus,Anguilla anguilla)

The catalytic activity of CYP1A isoforms and the effect of mammalian CYP1A-specific inhibitors in liver S9 fractions were studied in an agnathan (River lamprey, Lampetra fluviatilis, 30–33 cm) and in two species of teleost fish (European flounder, Pleuronectes flesus, 11–18 cm and common eel, Anguilla anguilla, 31–48 cm). Ethoxyresorufin O-deethylation (EROD), caffeine N-demethylation/C-oxidation and phenacetin O-deethylation (POD) activity increased 3–4-fold in flounders and 17–46-fold in eels, 5 days after fish were injected (i.p.) with 100 mg kg⁻¹ benzo(a)pyrene (B[a]P). In lampreys, basal EROD activity was very low and no increase in activity was observed following exposure to B[a]P. While the apparent Michaelis constant (K_m) for each assay showed only small changes after B[a]P injection, maximum reaction velocity (V_max) values increased by up to 19- and 84-fold for EROD activity, 4- and 35-fold for caffeine-related metabolism and 4- and 19-fold for POD activity in flounders and eels, respectively. The mammalian CYP1A2 inhibitor furafylline (50 μM–1 mM) reduced activity in the EROD, caffeine and POD assays to 65, 21 and 20% of control values in flounders and to 85, 10 and 5% of control values in eels, respectively. By contrast, low concentrations (0.025–0.050 μM) of the mammalian CYP1A1 inhibitor ellipticine completely abolished EROD activity, but had no effect (up to 1 mM) on caffeine metabolism or POD activity in either species. While the inhibitor studies strongly suggest that two separate enzymes are present in flounders and eels, the monophasic Michaelis–Menten kinetics obtained in all the assays imply that only a single CYP1A protein is present that has substrate and inhibitor specificities characteristic of both mammalian CYP1A1 and CYP1A2 isoforms.     

Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress–induced hepatosteatosis

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1^S724A/S724A) and investigated the importance of phosphorylation at Ser⁷²⁴ within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser⁷²⁴ exacerbated ER stress–induced hepatic steatosis in tunicamycin-treated Ern1^S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer–binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser⁷²⁴ of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.     

Comparative xenobiotic metabolism capacities and pesticide sensitivity in adults of Solea solea and Solea senegalensis

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Ecnomus of Madagascar,species without modified spur (Trichoptera: Ecnomidae)

The 11 Malagasy species of the genus Ecnomus McLachlan, 1864 that do not belong to the psychomyiellodes species group are studied. One of them (Ecnomus salgiel Malicky, 2015) was recently described from Nosy Be Island. Ten are new and described: Ecnomus felix n. sp., E. dobignyi n. sp., E. matsaboryi n. sp., E. granjoni n. sp., E. commersoni n. sp., E. menavavae n. sp., E. ikopaensis n. sp., E. rubermortis n. sp., E. evidens n. sp., and E. tendroensis n. sp. All are endemic to Madagascar. Their distributions on the island are briefly described according to ecological data.