PMC:3625153 / 27383-35493 JSONTXT

{"project":"2_test","denotations":[{"id":"23593491-14907713-88440278","span":{"begin":3491,"end":3493},"obj":"14907713"},{"id":"23593491-15716106-88440279","span":{"begin":5624,"end":5626},"obj":"15716106"},{"id":"23593491-17083914-88440286","span":{"begin":6053,"end":6055},"obj":"17083914"},{"id":"23593491-11808873-88440287","span":{"begin":7485,"end":7487},"obj":"11808873"},{"id":"T51532","span":{"begin":3491,"end":3493},"obj":"14907713"},{"id":"T14208","span":{"begin":5624,"end":5626},"obj":"15716106"},{"id":"T4767","span":{"begin":6053,"end":6055},"obj":"17083914"},{"id":"T52697","span":{"begin":7485,"end":7487},"obj":"11808873"}],"text":"Materials and Methods\n\nMaterials\nThe DetectX™ direct cyclic AMP enzyme immunoassay kit was purchased from Arbor Assays (US). The PepTag PKA assay kit was from Promega (Milan, Italy). Random primers and the RevertAid MulV reverse transcriptase were from Fermentas (Milan, Italy). The ChargeSwitch total RNA cell kit and the Fast Sybr Green reaction mix were from Life Technologies (Milan, Italy). Fluoxetine (FX), propranolol (PROP), noradrenaline (NOR), serotonin (5-HT), forskolin (FSK), dibutyryl cAMP (dbcAMP), H89 (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), protease inhibitor cocktail (P8340), and all other reagents were from Sigma Aldrich (Milan, Italy).\n\nMussel handling\nMytilus galloprovinicialis (4 to 6 cm in length) were obtained from a government certified mussel farm (Cooperativa Copr.al.mo, Cesenatico, Italy). They were transferred to the laboratory in seawater tanks and acclimated for 3 days in aquaria containing 35-psu filtered seawater at 16°C with continuous aeration (\u003e90% oxygen saturation). Mussels were fed once a day with a commercial algal slurry (Koral, Xaqua).\n\nIn vivo exposure experiments\nThirty mussels per treatment (6 individuals per vessel) were exposed for 7 days to 0.3 ng/L PROP, to 0.3 ng/L FX, or to the mixture FX+PROP (0.3 ng/L+0.3 ng/L). A group of unexposed (control) mussels were maintained in parallel to the treatment groups. Seawater was renewed each day and the chemicals added as stock solutions prepared in distilled water. Mussels were fed once a day. At the end of the exposure period, haemolymph was extracted from the posterior adductor muscle of each mussel using a sterile 1-mL syringe, centrifuged at 800×g for 10 min, snap frozen in liquid nitrogen and stored at −80°C.\n\nHaemocyte preparation and in vitro experiments\nHemolymph was extracted from the posterior adductor muscle of a number of individuals using a sterile 1-mL syringe then pooled to obtain the total volume required for each experiment. Hemolymph was then plated in 12-well plates (1 mL/well), and haemocytes were allowed settled for 1 h at 16°C in the dark. Cell attachment to the bottom of the well was checked microscopically. The medium was then removed, and cells were washed twice with 35-psu sterile artificial seawater (ASW). Control cells were incubated with 1 mL ASW, whereas 1 mL ASW containing the tested chemicals at the selected concentrations was added to the experimental wells. FSK and H89 were added to ASW from concentrated stock solutions prepared in dimethylsulphoxide (DMSO). In all cases, DMSO final concentration was 0.01% v/v and it did not significantly affect the biological endpoints analysed (data not shown). Experimental conditions (time of incubation and agonist/modulator concentrations) were assessed in preliminary trials to ensure significant evaluations of mRNA expressions along with cell signaling mediators. All incubations were carried out at 16°C in the dark. Each treatment consisted of 3 independent experiments, and each experimental trial consisted of 3 replicates for each biological endpoint (N = 3).\n\ncAMP assay\nHaemocytes were lysed in 65% ethanol for 1 h at 4°C. Lysates were centrifuged (2000×g for 15 min at 4°C), and the collected supernatants were dried under a stream of nitrogen. Contents of cAMP were assessed using the DetectX™ direct cyclic AMP enzyme immunoassay kit according to the manufacture's protocol. Total protein content was assessed using the Lowry method [55]. cAMP levels were expressed as pmol/mg protein.\n\nPKA activity assay\nHaemocytes were lysed in cold PKA extraction buffer containing 25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol and 50-fold diluted proteinase inhibitor cocktail. Supernatants were assayed for PKA activity using the non-radioactive PepTag PKA assay kit with dye-labeled Kemptide as a substrate according to manufacturer's protocol. Results were normalized to the total protein content and expressed as fold change vs control.\n\nQuantitative Real-Time PCR Analysis of ABCB and 5-HT1 mRNA Expressions\nTotal RNA was extracted from control and treated haemocytes using the ChargeSwitch total RNA cell kit according to the manufacturer's protocol. RNA concentration and quality were verified by UV spectroscopy and electrophoresis using a 1.2% agarose gel under denaturing conditions. First strand cDNA for each sample was synthesized from 1 µg total RNA in the presence of 250 ng random primers and 200 units RevertAid MulV reverse transcriptase following the manufacture's protocol.\nReal-time PCR reactions were performed in duplicate, in a final volume of 10 µL containing 5 µL Fast Sybr Green reaction mix with ROX, 2 µL diluted cDNA, and 0.2 µM specific primers (Table 1). A control lacking cDNA template was included in the real-time PCR analysis to determine the specificity of target cDNA amplification. Amplification was detected with a StepOne real time PCR system apparatus (Life Technologies, Milan, Italy) using a standard “fast mode” thermal protocol. For each target mRNA, melting curves, gel pictures and sequences were analysed in order to verify the specificity of the amplified products and the absence of artifacts. The amplification efficiency of each primer pair was calculated using a dilution series of cDNA (Table 1). A normalization factor, calculated using the geNorm software [56] and based on the expression levels of the best performing reference transcripts in the haemocyte samples, was used for accurate normalization of real-time PCR data. A set of suitable reference genes were selected from the literature [57]–[60] and are listed in Table 1; amongst these, the most stable reference genes used for normalization in haemocytes subjected to the different treatments were 18S rRNA and EF-α1 (Table 1).\n10.1371/journal.pone.0061634.t001 Table 1 List of primers used in real time PCR analyses. * primers were constructed basing on a partial sequence encoding an ABCB gene product from M. galloprovincialis (GenBank Ac Numb EF057747; [50]). A WWW-based database search using the BLAST program at NCBI found that this partial sequence showed a 65.17% and 64.93% nucleotide sequence identity with the human ABCB1 (GenBank Ac. Numb. NM_000927) and ABCB4 (GenBank Ac. Numb. NM_01884) gene sequences, respectively, while sequence identities lower or close to 50% were found with other human ABCB subtypes. Therefore, to avoid misleading information, the Pgp encoding gene from mussel investigated in this study will be referred to as ABCB gene. Relative expression of target genes in comparison with those of the reference genes was calculated by a comparative Ct method [61] using the StepOne software tool (Life Technologies, Milan, Italy). Data were finally reported as normalized relative expression (fold change) with respect to control samples.\n\nComputational analysis of the promoter region of ABCB genes from mussels\nTwo full length sequences encoding ABCB genes from Mytilus californianus (GenBank Ac. Numb. EF52141) and M. galloprovincialis (GenBank Ac. Numb. FM999809) were retrieved from the GenBank database. The ORF Finder Tool at the NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify the putative 5′-untraslated regions of both sequences. Sequence of the human ABCB1 promoter in the same region is reported for comparison (GenBank Ac. Numb. NM000927). Regulatory elements in the 5′-untranlasted regions were identified with Alibaba2 [62], MATCHTM [63] and the Transcription Element Search System (TESS) [64].\n\nStatistical analysis\nReal time PCR data were evaluated with the REST software [65] that uses a randomisation test with a pairwise reallocation to assess the statistical significance of the differences in expression between each treatment-exposure group and the control. cAMP levels and PKA activities were analysed using the SigmaStat statistical package. Significant differences between samples were determined using one-way ANOVA followed by the multiple comparison Bonferroni's test. In any case, statistical difference was accepted when p\u003c0.05. "}

{"project":"NEUROSES","denotations":[{"id":"T5","span":{"begin":757,"end":763},"obj":"PATO_0000122"},{"id":"T6","span":{"begin":1008,"end":1018},"obj":"PATO_0000689"},{"id":"T7","span":{"begin":1034,"end":1040},"obj":"CHEBI_15379"},{"id":"T8","span":{"begin":1034,"end":1040},"obj":"CHEBI_25805"}],"text":"Materials and Methods\n\nMaterials\nThe DetectX™ direct cyclic AMP enzyme immunoassay kit was purchased from Arbor Assays (US). The PepTag PKA assay kit was from Promega (Milan, Italy). Random primers and the RevertAid MulV reverse transcriptase were from Fermentas (Milan, Italy). The ChargeSwitch total RNA cell kit and the Fast Sybr Green reaction mix were from Life Technologies (Milan, Italy). Fluoxetine (FX), propranolol (PROP), noradrenaline (NOR), serotonin (5-HT), forskolin (FSK), dibutyryl cAMP (dbcAMP), H89 (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), protease inhibitor cocktail (P8340), and all other reagents were from Sigma Aldrich (Milan, Italy).\n\nMussel handling\nMytilus galloprovinicialis (4 to 6 cm in length) were obtained from a government certified mussel farm (Cooperativa Copr.al.mo, Cesenatico, Italy). They were transferred to the laboratory in seawater tanks and acclimated for 3 days in aquaria containing 35-psu filtered seawater at 16°C with continuous aeration (\u003e90% oxygen saturation). Mussels were fed once a day with a commercial algal slurry (Koral, Xaqua).\n\nIn vivo exposure experiments\nThirty mussels per treatment (6 individuals per vessel) were exposed for 7 days to 0.3 ng/L PROP, to 0.3 ng/L FX, or to the mixture FX+PROP (0.3 ng/L+0.3 ng/L). A group of unexposed (control) mussels were maintained in parallel to the treatment groups. Seawater was renewed each day and the chemicals added as stock solutions prepared in distilled water. Mussels were fed once a day. At the end of the exposure period, haemolymph was extracted from the posterior adductor muscle of each mussel using a sterile 1-mL syringe, centrifuged at 800×g for 10 min, snap frozen in liquid nitrogen and stored at −80°C.\n\nHaemocyte preparation and in vitro experiments\nHemolymph was extracted from the posterior adductor muscle of a number of individuals using a sterile 1-mL syringe then pooled to obtain the total volume required for each experiment. Hemolymph was then plated in 12-well plates (1 mL/well), and haemocytes were allowed settled for 1 h at 16°C in the dark. Cell attachment to the bottom of the well was checked microscopically. The medium was then removed, and cells were washed twice with 35-psu sterile artificial seawater (ASW). Control cells were incubated with 1 mL ASW, whereas 1 mL ASW containing the tested chemicals at the selected concentrations was added to the experimental wells. FSK and H89 were added to ASW from concentrated stock solutions prepared in dimethylsulphoxide (DMSO). In all cases, DMSO final concentration was 0.01% v/v and it did not significantly affect the biological endpoints analysed (data not shown). Experimental conditions (time of incubation and agonist/modulator concentrations) were assessed in preliminary trials to ensure significant evaluations of mRNA expressions along with cell signaling mediators. All incubations were carried out at 16°C in the dark. Each treatment consisted of 3 independent experiments, and each experimental trial consisted of 3 replicates for each biological endpoint (N = 3).\n\ncAMP assay\nHaemocytes were lysed in 65% ethanol for 1 h at 4°C. Lysates were centrifuged (2000×g for 15 min at 4°C), and the collected supernatants were dried under a stream of nitrogen. Contents of cAMP were assessed using the DetectX™ direct cyclic AMP enzyme immunoassay kit according to the manufacture's protocol. Total protein content was assessed using the Lowry method [55]. cAMP levels were expressed as pmol/mg protein.\n\nPKA activity assay\nHaemocytes were lysed in cold PKA extraction buffer containing 25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol and 50-fold diluted proteinase inhibitor cocktail. Supernatants were assayed for PKA activity using the non-radioactive PepTag PKA assay kit with dye-labeled Kemptide as a substrate according to manufacturer's protocol. Results were normalized to the total protein content and expressed as fold change vs control.\n\nQuantitative Real-Time PCR Analysis of ABCB and 5-HT1 mRNA Expressions\nTotal RNA was extracted from control and treated haemocytes using the ChargeSwitch total RNA cell kit according to the manufacturer's protocol. RNA concentration and quality were verified by UV spectroscopy and electrophoresis using a 1.2% agarose gel under denaturing conditions. First strand cDNA for each sample was synthesized from 1 µg total RNA in the presence of 250 ng random primers and 200 units RevertAid MulV reverse transcriptase following the manufacture's protocol.\nReal-time PCR reactions were performed in duplicate, in a final volume of 10 µL containing 5 µL Fast Sybr Green reaction mix with ROX, 2 µL diluted cDNA, and 0.2 µM specific primers (Table 1). A control lacking cDNA template was included in the real-time PCR analysis to determine the specificity of target cDNA amplification. Amplification was detected with a StepOne real time PCR system apparatus (Life Technologies, Milan, Italy) using a standard “fast mode” thermal protocol. For each target mRNA, melting curves, gel pictures and sequences were analysed in order to verify the specificity of the amplified products and the absence of artifacts. The amplification efficiency of each primer pair was calculated using a dilution series of cDNA (Table 1). A normalization factor, calculated using the geNorm software [56] and based on the expression levels of the best performing reference transcripts in the haemocyte samples, was used for accurate normalization of real-time PCR data. A set of suitable reference genes were selected from the literature [57]–[60] and are listed in Table 1; amongst these, the most stable reference genes used for normalization in haemocytes subjected to the different treatments were 18S rRNA and EF-α1 (Table 1).\n10.1371/journal.pone.0061634.t001 Table 1 List of primers used in real time PCR analyses. * primers were constructed basing on a partial sequence encoding an ABCB gene product from M. galloprovincialis (GenBank Ac Numb EF057747; [50]). A WWW-based database search using the BLAST program at NCBI found that this partial sequence showed a 65.17% and 64.93% nucleotide sequence identity with the human ABCB1 (GenBank Ac. Numb. NM_000927) and ABCB4 (GenBank Ac. Numb. NM_01884) gene sequences, respectively, while sequence identities lower or close to 50% were found with other human ABCB subtypes. Therefore, to avoid misleading information, the Pgp encoding gene from mussel investigated in this study will be referred to as ABCB gene. Relative expression of target genes in comparison with those of the reference genes was calculated by a comparative Ct method [61] using the StepOne software tool (Life Technologies, Milan, Italy). Data were finally reported as normalized relative expression (fold change) with respect to control samples.\n\nComputational analysis of the promoter region of ABCB genes from mussels\nTwo full length sequences encoding ABCB genes from Mytilus californianus (GenBank Ac. Numb. EF52141) and M. galloprovincialis (GenBank Ac. Numb. FM999809) were retrieved from the GenBank database. The ORF Finder Tool at the NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify the putative 5′-untraslated regions of both sequences. Sequence of the human ABCB1 promoter in the same region is reported for comparison (GenBank Ac. Numb. NM000927). Regulatory elements in the 5′-untranlasted regions were identified with Alibaba2 [62], MATCHTM [63] and the Transcription Element Search System (TESS) [64].\n\nStatistical analysis\nReal time PCR data were evaluated with the REST software [65] that uses a randomisation test with a pairwise reallocation to assess the statistical significance of the differences in expression between each treatment-exposure group and the control. cAMP levels and PKA activities were analysed using the SigmaStat statistical package. Significant differences between samples were determined using one-way ANOVA followed by the multiple comparison Bonferroni's test. In any case, statistical difference was accepted when p\u003c0.05. "}