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PubMed2005

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Clinical Laboratory Techniques↗

PubMed1998

Amino acid substitutions in PilD, a bifunctional enzyme of Pseudomonas aeruginosa. Effect on leader peptidase and N-methyltransferase activities in vitro and in vivo.

Subunits of type IV pili and a subset of proteins of the type II extracellular protein secretion apparatus undergo two consecutive post-translational modifications: leader peptide cleavage, followed by methylation of the newly created N-terminal amino acid. These two reactions are carried out by a single bifunctional enzyme encoded in Pseudomonas aeruginosa by the pilD gene. Properties of PilD mutants at positions Gly95 and/or Lys96 which were differentially affected in leader peptidase and N-methyltransferase function were characterized. None of the single amino acid substitutions showed a significant alteration in their ability to cleave the prepilin leader peptide; however, two double mutants did exhibit a modest reduction in the efficiency of cleavage. In contrast, a significant decrease of N-methyltransferase activity was detected in PilD having substitutions at Gly95. Mutants with substitutions at position Lys96 showed a variable effect on N-methyltransferase activity with an apparent requirement for any charged amino acid at this position. Absence of N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional pili. Moreover, pilin monomers isolated from P. aeruginosa expressing PilD with Gly95 substitutions were not methylated. Although complete methylation does not appear to be absolutely required for pilus assembly in P. aeruginosa, this modification may be important for pilus function in its natural habitat.

Amino Acid Sequence↗

PubMed2006

Aquatic toxicity of nine aircraft deicer and anti-icer formulations and relative toxicity of additive package ingredients alkylphenol ethoxylates and 4,5-methyl-1H-henzotriazoles.

Characterization of the effects of aircraft deicer and anti-icer fluid (ADAF) runoff on aquatic organisms in receiving streams is a complex issue because the identities of numerous toxic additives are proprietary and not publicly available. Most potentially toxic and endocrine disrupting effects caused by ADAF are due to the numerous additive package ingredients which vary among manufacturers and types of ADAF formulation. Toxicity investigations of nine ADAF formulations indicate that endpoint concentrations for formulations of different manufacturers are widely variable. Type IV ADAF (anti-icers) are more toxic than Type I (deicers) for the four organisms tested (Vibrio fischeri, Pimephales promelas, Ceriodaphnia dubia, and Selenastrum capricornutum). Acute toxicity endpoint concentrations ranged from 347 to 7700 mg/L as ADAF for Type IV and from 1550 to 45,100 mg/L for Type I formulations. Chronic endpoint concentrations ranged from 70 to 1300 mg/L for Type IV and from 37 to 18,400 mg/L for Type I formulations. Alkylphenol ethoxylates and tolyltriazoles are two known classes of additives. Nonylphenol, nonylphenol ethoxylates, octylphenol, octylphenol ethoxylates, and 4,5-methyl-1H-benzotriazoles were quantified in the nine ADAF formulations, and toxicity tests were conducted with nonylphenol ethoxylates and 4,5-methyl-1H-benzotriazoles. Toxicity units computed for glycol and these additives, with respect to toxicity of the ADAF formulations, indicate that a portion of ADAF toxicity can be explained by the known additives and glycols, but much of the toxicity is due to unidentified additives.

Aircraft↗

PubMed1990

Modulating role of dopamine on anesthetic requirements.

The influence of dopamine on halothane anesthetic requirements was determined in mice. Halothane anesthetic requirement was defined as the minimum anesthetic concentration (MAC) that prevented 50% animals from moving in response to a supramaximal stimulus. Levodopa (L-DOPA) dose-dependently decreased halothane MAC to a maximum of 49% of control; over the same dose range L-DOPA increased striatal dopamine nearly 4-fold. The MAC-reducing effect of L-DOPA was attenuated by selective antagonism of the D2 dopamine receptor with YM-09151-2 while selective blockade of the D1 dopamine receptor with SCH-23390 did not alter L-DOPA's effect on the MAC for halothane. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreased striatal dopamine by 82% and increased the MAC for halothane by 27%. Repletion of striatal dopamine with L-DOPA, in MPTP-treated mice, restored the MAC for halothane back to the control state. The regression line derived from the plot of halothane MAC versus striatal dopamine content shows a highly significant correlation between the two variables (r2 = 0.94). These are the first results to suggest that anesthetic requirements can be modulated directly and precisely by increasing or decreasing the content of a single neurotransmitter in the central nervous system.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2003

Polymorphisms in the human monomethylarsonic acid (MMA V) reductase/hGSTO1 gene and changes in urinary arsenic profiles.

Large interindividual variability in urinary arsenic profiles, following chronic inorganic arsenic exposure, is well-known in humans. To understand this variability, we studied the relationship between polymorphisms in the gene for human monomethylarsonic acid (MMA(V)) reductase/hGSTO1 and the urinary arsenic profiles of individuals chronically exposed to arsenic in their drinking water. To ensure that we did not overlook rare polymorphisms, not included in the public databases, we amplified and sequenced all six exons of the gene and their flanking regions, using DNA isolated from peripheral blood samples of 75 subjects, living in the vicinity of Torreon, Mexico. Four groups, based on the levels of arsenic (9-100 microg/L) in their drinking water, were studied. We identified six novel polymorphisms and two reported previously. The novel polymorphisms were a three base pair deletion (delGGC) in the first intron; a G > C transversion, leading to a serine-to-cysteine substitution at amino acid 86; a G > T transversion and a A > T transversion in intron 5; a G > A transition resulting in glutamate-to-lysine substitution in amino acid 208; and a C > T transition producing an alanine-to-valine substitution in amino acid 236. Two subjects displayed significant differences in patterns of urinary arsenic; they had increased levels of urinary inorganic arsenic and reduced levels of methylated urinary arsenic species as compared to the rest of the study population. These two subjects had the same unique polymorphisms in hGSTO1 in that they were heterozygous for E155del and Glu208Lys. The identified SNPs may be one of the reasons for the large interindividual variability in the response of humans to chronic inorganic arsenic exposure. The findings suggest the need for further studies to identify unambiguously specific polymorphisms that may account for interindividual variability in the human response to chronic inorganic arsenic exposure.

Adolescent↗

PubMed2001

Cerebral vasoconstriction produced by vasopressin in conscious goats: role of vasopressin V(1) and V(2) receptors and nitric oxide.

To examine the role of vasopressin V(1) and V(2) receptors, nitric oxide and prostanoids in the cerebrovascular effects of arginine vasopressin, cerebral blood flow was electromagnetically measured in awake goats. In 16 animals, vasopressin (0.03 - 1 microg), injected into the cerebral circulation, caused increments of resting cerebrovascular resistance which ranged from 18% (0.03 microg, P<0.01) to 79% (1 microg, P<0.01). Desmopressin (0.03 - 1 microg, four goats) did not affect significantly cerebrovascular resistance. The cerebrovascular resistance increases by vasopressin were reduced significantly by the antagonist for vasopressin V(1) receptors d(CH(2))(5)Tyr(Me)-AVP in a rate depending way (five (six goats) and 15 (four goats) microg min(-1)), and by the mixed antagonist for vasopressin V(1) and V(2) receptors desGly-d(CH(2))(5)-D-Tyr(Et)Val-AVP (5 microg min(-1), four goats), and they were not significantly affected by the antagonist for vasopressin V(2) receptors d(CH(2))(5), D-Ile(2), Ile(4)-AVP (5 microg min(-1), four goats). The inhibitor of nitric oxide synthesis N(w)-nitro-L-arginine methyl ester (L-NAME, 47 mg kg(-1) i.v., five goats) augmented cerebrovascular resistance by 130% (P<0.01), and for 24 h after this treatment the cerebrovascular effects of vasopressin were potentiated. The inhibitor of cyclo-oxygenase meclofenamate (6 mg kg(-1) i.v., five goats) did not modify significantly resting haemodynamic variables measured or the cerebrovascular effects of vasopressin. Therefore, the vasopressin-induced cerebral vasoconstriction may be mediated by vasopressin V(1) receptors, without involvement of vasopressin V(2) receptors, and may be modulated by nitric oxide but not by prostanoids.

Animals↗

PubMed2004

Nitrogen oxide blockade does not aggravate the endothelin-1-induced myocardial ischemia and release of purine metabolites from the dog heart.

Increased intrapericardial levels of endothelin-1 (ET-1) induce myocardial ischemia and concomitant release of the purine metabolites adenosine (ADO), inosine (INO) and hypoxanthine (HXA) into the pericardial fluid. However, the potential modulatory role of nitrogen monoxide in compensating the ET-1-induced ischemic stress is not fully elucidated. The pericardial elevations of purine metabolite concentrations in the pericardial fluid after ET-1 administration (150 pmol/kg intrapericardially) were measured in the in situ dog heart with (n = 6) or without (n = 5) systemic nitrogen monoxide synthase blockade (30 mg/kg (G)-nitro-L-arginine methyl ester, followed by 6 mg/min intravenously). After control sampling, three consecutive pericardial infusate samples (ET1, ET2, ET3) were obtained for purine metabolite determinations (high-performance liquid chromatography-ultraviolet). It was found that intrapericardial ET-1 elevated the pericardial purine metabolite concentrations significantly in both groups. No significant differences were detected between the control and (G)-nitro-L-arginine methyl ester-treated groups in ischemic changes of pericardial ADOmax (+3.27 +/- 1.13 microM versus +1.84 +/- 0.56 microM), INOmax (+15.21 +/- 2.3 microM versus +12.09 +/- 4.04 microM) and HXAmax (+16.34 +/- 2.98 microM versus +17.09 +/- 5.22 microM) levels and in the maximal ST elevations (0.43 +/- 0.05 mV versus 0.61 +/- 0.08 mV). The hemodynamic variables did not change with ET-1 administration. In conclusion, systemic nitrogen monoxide synthase blockade does not aggravate the ET-1-induced acute myocardial ischemia and the release of purine metabolites, suggesting that endogenous nitrogen monoxide is not a supplementary factor to purine metabolites in this type of coronary adaptive responses.

Adaptation, Physiological↗

PubMed2025

The early injected genomic region determines sensitivity to Type I restriction-modification defence against Autographiviridae phages.

Bacteriophages must evade bacterial defences to establish successful infections. Type I restriction-modification (RM) systems recognize specific DNA motifs and degrade unmethylated foreign DNA, restricting phage replication. In this study, we detected that Marinomonas mediterranea MMB-2 uses a Type I RM system (Mme2I) to protect against several new phages in the Murciavirus genus within the Autographiviridae family. Whole-genome sequencing and methylation analysis revealed a DNA sequence motif methylated in M. mediterranea MMB-2, which is also present in the phages. Phages lacking the motif within the leading, first injected, region of their genomes, either natural isolates or escape mutants of sensitive phages, successfully infect M. mediterranea MMB-2, despite the presence of the recognition motif elsewhere in their genomes. These results highlight the importance of considering RM motif locations when predicting avoidance of restriction sites as escape mechanisms from RM systems. Additionally, our findings indicate an important role for RM systems in specifically influencing the organization of the leading injected regions of phage genomes, which are highly variable and often encode diverse anti-defence systems.

Genome, Viral↗

PubMed2000

Confounding variables in the environmental toxicology of arsenic.

Arsenic is one of the most important global environmental toxicants. For example, in regions of West Bengal and Inner Mongolia, more than 100000 persons are chronically exposed to well water often strongly contaminated with As. Unfortunately, a toxicologically safe risk assessment and standard setting, especially for long-term and low-dose exposures to arsenic, is not possible. One reason is that the key mechanism of arsenic's tumorigenicity still is not elucidated. Experimental data indicate that either DNA repair inhibition or DNA methylation status alteration may be causal explanations. Moreover, when comparing epidemiological data, it cannot be ruled out that the susceptibility to arsenic's carcinogenicity may be different between Mexican and Taiwanese people. Some other studies indicate that some Andean populations do not develop skin cancer after long-term exposure to As. It is not known yet how this resistance could be mediated. Finally, the situation is even more complicated when taking into consideration that there are several compounds suspected to modulate the chronic environmental toxicity of arsenic, variables that may either enhance or suppress the in vivo genotoxicity and carcinogenicity of the metalloid. Among them are nutritional factors like selenium and zinc as well as drinking water co-contaminants like antimony. Further, yet unidentified factors influencing the body burden and/or the excretion of arsenic are possibly prevailing: preliminary data from own human biomonitoring studies showed a peaking of As in urine samples of non-exposed people which was not caused by elevated exposure to As through seafood consumption. The relevance of these putative confounding variables cannot be finally evaluated yet. Further experimental as well as epidemiological studies are needed to answer these questions. This would help to conduct a toxicologically improved risk assessment, especially for low-dose and long-term exposures to arsenic.

Arsenic Poisoning↗

PubMed2003

Identification of MeCP2 mutations in a series of females with autistic disorder.

Rett disorder and autistic disorder are both pervasive developmental disorders. Recent studies indicate that at least 80% of Rett Disorder cases are caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Since there is some phenotypic overlap between autistic disorder and Rett disorder, we analyzed 69 females clinically diagnosed with autistic disorder for the presence of mutations in the MeCP2 gene. Two autistic disorder females were found to have de novo mutations in the MeCP2 gene. These data provide additional evidence of variable expression in the Rett disorder phenotype and suggest MeCP2 testing may be warranted for females presenting with autistic disorder.

Adolescent↗

PubMed1998

Roles of neuroactive amino acids in ammonia neurotoxicity.

Many neurologic disorders are related to congenital or acquired hyperammonemia (HA). Advanced symptoms of HA range from seizures in acute stages to stupor and coma in more chronic conditions, manifesting variable imbalance between the inhibitory and excitatory neurotransmission. Evidence obtained with the use of experimental HA models suggests that acute neurotoxic effects of ammonia are mediated by overactivation of ionotropic glutamate (GLU) receptors, mainly the N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree the KA/AMPA receptors. NMDA receptor-mediated neurotoxicity may be potentiated by impaired control of their function by metabotropic GLU receptors, which are inactivated by ammonia. Prolonged overactivation of the NMDA receptors upon extended ammonia exposure causes their downregulation. The GLU receptor changes may be related to their excessive exposure to extrasynaptic GLU. Ammonia promotes GLU accumulation in the extrasynaptic space by enhancing its release from neurons, and/or by decreasing its reuptake to the nerve endings and astrocytes, where the effect results from inactivation (downregulation) of the astrocytic glutamate transporter GLT1. Excitotoxic effects of ammonia are augmented by increased synthesis of nitric oxide (NO), which is associated with NMDA receptor activation and/or increased synaptic transport of arginine (ARG). A shift toward neural inhibition is promoted by positive modulation of the gamma-aminobutyric acid (GABA)ergic tone resulting from excessive accumulation in the brain of endogenous central benzodiazepine receptor agonists, and from upregulation of astrocytic peripheral benzodiazepine receptors leading to elevated levels of prognenelone-derived neurosteroids, which positively modulate the GABA(A) receptor complex. Inhibitory neurotransmission may also be favored by enhanced release from astrocytes of an inhibitory amino acid, taurine.

Ammonia↗

PubMed2002

1,8-bis(tetramethylguanidino)naphthalene (TMGN): a new, superbasic and kinetically active "proton sponge".

1,8-Bis(tetramethylguanidino)naphthalene (TMGN, 1) is a new, readily accessible, and stable "proton sponge" with an experimental pK(BH(+)) value of 25.1 in MeCN, which is nearly seven orders of magnitude higher in basicity than the classical proton sponge 1,8-bis(dimethylamino)-naphthalene (DMAN). Because of the sterically less crowded character of the proton-accepting sp(2)-nitrogen atoms, TMGN also has a higher kinetic basicity than DMAN, which is shown by time-resolved proton self-exchange reactions. TMGN is more resistant to hydrolysis and is a weaker nucleophile towards the alkylating agent EtI in comparison to the commercially available guanidine 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD). Crystal structures of the free base, of the mono- and bisprotonated base were determined. The dynamic behavior of all three species in solution was investigated by variable-temperature (1)H NMR experiments. DeltaG (++) values obtained by spectra simulation reveal a concerted mechanism of rotation about the C-N bonds of the protonated forms of TMGN.

Journal Article↗

PubMed2000

Analysis of colistin sulfate by capillary zone electrophoresis with cyclodextrins as additive.

A method for the quantitative analysis of colistin sulfate by capillary zone electrophoresis is described. Since colistin components have five free amino groups, they tend to adsorb onto the capillary wall and cause peak tailing. It was found that triethanolamine (TEA)-phosphate buffer at pH 2.5 was useful to reduce such adsorption. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found necessary for selectivity enhancement. In order to optimize the separation parameters and predict the method robustness, a central composite design was performed including three variables, namely concentration of M-beta-CD, TEA, and IPA. The effects of capillary length and applied voltage on separation were also investigated. The optimal conditions established were: 140 mM TEA-phosphate buffer containing 5 mM M-beta-CD and 6% v/v IPA, a capillary with 55 cm total length (50 microm inner diameter, 47 cm from inlet to detection window) and 24 kV applied voltage. The method was found to be robust when the variables were changed in the following range: 4-6 mM M-beta-CD, 5-7% v/v IPA, and 130-150 mM TEA. Further, the linearity, limit of detection (LOD), and limit of quantitation (LOQ), as well as repeatability for both colistin A and B were examined and three commercial samples were quantitatively analyzed.

1-Propanol↗

PubMed2002

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozygosity in locally advanced laryngeal squamous cell carcinoma.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

Base Sequence↗

PubMed2001

Inactivation of O6-alkylguanine-DNA alkyltransferase by 8-substituted O6-benzylguanine analogs in mice.

PURPOSE: The purpose of this study was to determine the usefulness of various 8-substituted O6-benzylguanine (BG) analogs as modulators of the DNA repair protein. O6-alkylguanine-DNA alkyltransferase (AGT). More specifically, the degree of inactivation of AGT in mouse brain, liver, kidney and tumor by O6-benzyl-8-oxoguanine (8-oxoBG), 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-bromoBG) and O6-benzyl-8-trifluoromethylguanine (8-tfmBG) was compared to inactivation by BG, a modulator in phase II clinical trials. BG is converted rapidly to 8-oxoBG in rodents, monkeys and humans. It was reasoned that 8-substituted analogs of BG would exhibit different pharmacological properties compared to BG which could influence tissue bioavailability and, thus, the extent of AGT inactivation in vivo. We compared the tissue distribution of these agents and AGT activity following administration of the 8-substituted analogs. MATERIALS AND METHODS: At various time points up to 24 h after i.p. administration of the BG analogs, tissues (i.e. brain, liver, kidney), A549 lung tumor xenografts (i.p.) or D456 brain tumor xenografts (i.c.) were harvested from athymic nude mice for AGT analysis. AGT activity was quantified in tissue extracts using a biochemical assay with [3H]methylated DNA as a substrate. In addition, concentrations of BG and 8-oxoBG were determined by HPLC with fluorescence detection in mouse tissues following administration of drug. RESULTS: Each of the 8-substituted analogs of BG demonstrated variable AGT inactivation capabilities that were comparable to or better than those of BG especially in kidney and brain tissues. There was a more pronounced depletion of AGT inactivation in brain and D456 brain tumor xenografts following administration of BG compared to 8-oxoBG that could be explained by a much greater concentration of AGT-inactivating drug (BG plus the metabolite 8-oxoBG for mice treated with BG versus 8-oxoBG for mice treated with 8-oxoBG) present in these tissues. The AUCs for brain, kidney and liver were 3.2, 6.9 and 1 1.8 times greater for BG than for 8-oxoBG. CONCLUSIONS: 8-substituted analogs of BG possess unique AGT-inactivation profiles in vivo that are different from that of BG. The AGT-inhibitory activities of BG and its major metabolite, 8-oxoBG, are related to tissue disposition of both drugs.

Animals↗

PubMed2005

Water sorption-desorption behaviour of methyl methacrylate-starch copolymers: effect of hydrophobic graft and drying method.

A new family of graft copolymers combining hydrophilic and hydrophobic components have recently been proposed as direct compression excipients. Copolymers were synthetised by free radical copolymerisation of starch derivatives with methyl methacrylate (MMA) and were alternatively dried by oven or freeze-drying techniques. The aim of this study was to investigate the water vapour sorption-desorption behaviour of these copolymers, focusing on the influence of variables such as the hydrophobic component and the drying process. Moisture sorption and desorption isotherms were measured at 25 degrees C and analysed according to GAB and Young-Nelson equations, which distinguish between different physical forms of moisture distribution. The Young-Nelson model gave the best fit to the experimental data. The results obtained showed that the presence of the acrylic component modified not only the total hygroscopicity of the copolymers, as compared with the original starch derivatives, but also the water distribution in the solid, which might have an important role in the effect of moisture content on copolymer characteristics. The main water-sorption mechanism seemed to be absorption into the copolymer particles structure, in agreement with their starching nature. In terms of water sorption-desorption characteristics, no marked differences were found between the two drying methods used.

Desiccation↗

PubMed2004

Molecular markers for animal biotechnology: sea bass (Dicentrarchus labrax, L.) HMG-CoA reductase mRNA.

Modern technologies may improve fish production and quality and, at the same time, reduce environmental impact with benefits on the public perception of the industry. To be economically profitable, these modern technologies request an increase of rearing density that, however, could affect fish welfare. With the aim to search for molecular biomarkers to describe fish welfare, we have recently compared gene expression of sea bass farmed at different population densities by differential display obtaining six bands differentially expressed. In this paper, we have cloned the mRNA corresponding to one of those differentially expressed bands obtaining a 3860-bp sequence with an ORF of 2664 bp. Its virtual translation originated a 887-aa polypeptide that, by comparison with the other sequences available in the public data bases, resulted to be the 3-hydroxil-3-methyl-glutaryl coenzyme A reductase (HMGCR). In sea bass, as for the other species, the N- and C-terminus portions are the most conserved and are linked by an hydrophilic region that appears to be quite variable. Due to its role in the synthesis of cholesterol, HMGCR mRNA could be a good biomarker for detecting fish welfare. For this reason, we also followed, by real-time PCR, its expression after crowding stress comparing it with mRNA levels of HSP 70 and 90: HMGCR mRNA resulted highly expressed in the fishes farmed at 100 kg/m(3).

Animals↗

PubMed2002

Stimulus generalization by fenfluramine in a quipazine-ketanserin drug discrimination is not dependent on indirect serotonin release.

The purpose of this study was to determine if animals trained to discriminate a serotonin2A (5-HT2A) receptor agonist from a 5-HT2A receptor antagonist would also be sensitive to alterations in serotonin neurotransmission brought about by 5-HT reuptake inhibitors and releasers. Previous work from our laboratory has shown that the quipazine-ketanserin discrimination is mediated solely by the 5-HT2A receptor, thus providing a behavioral continuum of 5-HT2A receptor function. Rats were trained to discriminate quipazine (0.35 mg/kg) from ketanserin (1.0 mg/kg) on a variable interval-30 schedule of reinforcement. Following acquisition, substitution tests were conducted with the training drug, quipazine, and agents that have been shown to alter the synaptic levels of 5-HT, including fenfluramine, norfenfluramine, 5-methoxy-6-methyl-2-aminoindan (MMAI) and fluoxetine. All compounds substituted, except fluoxetine. Antagonist tests with mianserin and MDL 100,907 indicated that fenfluramine's and MMAI's substitution for quipazine was mediated by the 5-HT2A receptor. Animals were pretreated with PCPA to determine whether 5-HT release or direct agonism mediated the discriminative stimulus effects of fenfluramine and MMAI. PCPA blocked the substitution of MMAI but not of fenfluramine for quipazine. Analysis of 3H-IP formation in cells showed that norfenfluramine dose-dependently stimulated phosphoinositide hydrolysis to levels similar to that of serotonin and quipazine. These results indicate that fenfluramine's substitution for quipazine in rats trained on a quipazine-ketanserin discrimination are due to direct agonism at the 5-HT2A receptor likely mediated by norfenfluramine, an active metabolite.

Animals↗