Acute toxicity of a commercial glyphosate formulation on European sea bass juveniles (Dicentrarchus labrax L.): gene expressions of heme oxygenase-1 (ho-1), acetylcholinesterase (AChE) and aromatases (cyp19a and cyp19b).

Acute toxicity of Roundup, a commercial glyphosate--based herbicide, was evaluated in a teleost marine fish, the European sea bass, after 96 h of exposure. The LC50 96-h value of Roundup was 529 mg/L. Juveniles (Dicentrarchus labrax L.) were exposed to a sublethal concentration (35% of the LC50, i.e. 193 mg/L) of Roundup for 96-h. The study of heme oxygenase-1 (ho-1) gene expression was performed in four tissues (liver, gills, brain and gonads) and highlighted the disruption of antioxidant defence system. Results showed that ho-1 mRNA levels in liver and gills significantly decreased (p<0.001 and p<0.01 respectively) in fish exposed to 193 mg/L of Roundup, whereas in brain and gonads, ho-1 mRNA level was not altered. The analysis of acetylcholinesterase expression was used to evaluate the overall neurotoxicity of the herbicide and aromatase genes to assess the alteration of the endocrine system. Results showed that AChE and cyp19b gene transcriptions significantly increased (p<0.01) in brain of sea bass, whereas aromatase gene expression (cyp19a) in gonads was not significantly altered. Our results showed complex tissue-specific transcriptional responses after 96 h of exposure to a sublethal concentration. All these disruptions confirmed the deleterious effects of this glyphosate-based herbicide in a marine species.

Effect of water temperature increase on HO-1 expression in European sea bass (Dicentrarchus labrax L.) tissues.

One of the most pertinent environmental factors influencing the marine organism life is temperature. It has been demonstrated that an increase of temperature is able to induce the synthesis of heat shock proteins (HSP). In this study we investigated the expression of HO-1 mRNA, also referred to as HSP32, in different tissues of European sea bass (Dicentrarchus labrax, L.) at several time points after increased temperature exposure (from 12degC to 30degC). Our results showed that HO-1 was not expressed in gills, heart, muscle and brain while it was expressed at a basal level in intestine. In liver, spleen and kidneys, HO-1 expression was influenced by temperature increases. In the spleen, we found a significant decrease of the HO-1 expression at the end of 4 weeks. In kidneys a very fast collapse of HO-1 expression level was recorded reaching null value as soon as one hour after exposure to 30degC. In liver, HO-1 expression increased from one hour of exposure to 30degC confirming HO-1 involvement to heat shock response in this organ. This increasing trend reached a 4.5-fold higher value than the initial level after 4 weeks.

Effects of water temperature increase and heavy metals contamination on WAP65 gene expression in sea bass (Dicentrarchus labrax) liver.

It has been previously demonstrated that "Warm temperature Acclimation-related 65 kD Protein" (WAP65) is involved in temperature acclimation, response to intoxication and infection, as well as in development. The expression of wap65-1 was investigated in the liver of European sea bass (Dicentrarchus labrax) during exposure to the increased temperature (from 12 deg C to 30 deg C) and during intoxication with four heavy metals: lead, cadmium, copper and zinc. Post temperature increase wap65 expression was highest after one hour at 30 deg C. After 1 to 4 weeks at 30 deg C wap65 transcript levels did not differ from the 12 deg C control group, similar to observations regarding the heat shock protein, hsp70. Upregulation of wap65 was detected after treatment (intoxication) with cadmium (0.5 µg/l). In contrast, a slight, but significant down regulation of wap65 was seen after copper (5 µg/l) intoxication. These data indicate that functional analyses of WAP65 are needed to understand the differential regulation of this gene by metals. The role of WAP65 may be similar to that of HSP70, which has generalized functions in responding to certain stressors and maintaining normal cell physiology.

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues.

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5' and 3' rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709bp and the coding sequence is flanked by a 67bp 5'-UTR and a 358bp 3'-UTR. The full-length cDNA sequence of S. aurata is 1599bp, and the coding sequence is flanked by a 48bp 5'-UTR and a 273bp 3'-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.

cDNA sequencing and expression analysis of Dicentrarchus labrax heme oxygenase-1.

The liver cDNA encoding heme oxygenase--1 (HO-1) was sequenced from European sea bass (Dicentrarchus labrax) (accession number no. EF139130). The HO-1 cDNA was 1250 bp in nucleotide length and the open reading frame encoded 277 amino acid residues. The deduced amino acid sequence of the European sea bass had 75% and 50% identity with the amino acid sequences of tetraodontiformes (Tetraodon nigroviridis and Takifugu rubripes) and human HO-1 proteins, respectively. A short hydrophobic transmembrane domain at the C--terminal region was found, and four histidine residues were highly conserved, including human his25 that is essential for HO catalytic activity. RT-PCR of mRNA from eight different European sea bass tissues revealed that, in a homeostatis state, the heme oxygenase--1 was abundant in the spleen and liver but not in the brain.

Sequence protein alignment with composition new evolutions (SPACne): a program for the identification of polypeptides using amino acid composition. A user friendly modification of SPAC.

SPAC (sequence protein alignment with composition) is a software that retrieve from protein or nucleic acid databases, the sequences corresponding to a protein or peptide whose only amino acid composition and molecular weight are known. By accurately matching a DNA or a protein sequence to candidate protein or peptide fragment, this software may be used as a fast and cheap method for protein characterization. This paper describes a modification of the SPAC software, SPAC new evolutions (SPACne), which enables a more efficient and user friendly method of protein identification using amino acids composition. SPACne is available online at the web site: http://bioweb.pasteur.fr/seqanal/interfaces/spacne.html.

RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation modulates the effects of interleukin-1beta on rat chondrocytes.

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.

SPAC: identification of polypeptides using their amino-acid composition.

We describe here new software able to retrieve from protein or nucleic acid databases, the sequence corresponding to a protein or peptide whose only amino acid composition and molecular weight are known. This algorithm is particularly devoted to the retrieval of partial sequences, a task that other available software performs poorly. Its accuracy for the attribution of a protein fragment to a sequence could represent an easy and economical first tool upstream the use of more sophisticated and expensive methods in proteomic research. SPAC (Sequence Protein Alignment with Composition) is a shareware available software on web site http://www.univ-tln.fr/ approximately grillas/SPAC/. This web site also presents help and a detailed description of the algorithm and interface.

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE: To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS: Freund's complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS: After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION: A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.

UDP-glucuronosyltransferase in the rat olfactory bulb: identification of the UGT1A6 isoform and age-related changes in 1-naphthol glucuronidation.

Xenobiotic glucuronidation represents a major metabolic protection of the brain against chemical aggressions at blood-brain interfaces. We previously observed that glucuronidation of 1-naphthol was very effective in olfactory bulb, which is a pathway for the entry of foreign molecules into the brain. In this work, we showed that 1-naphthol glucuronidation varied according to age. It was very high at birth, then decreased markedly in 3-month-old rats and increased again significantly during aging. By Western blot and reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the presence in the olfactory bulb of the UDP-glucuronosyltransferase (UGT) 1A6 isoform, which catalyzes the glucuronidation of phenols, such as 1-naphthol. Quantitative RT-PCR indicated that the mRNA levels encoding UGT1A6 did not significantly change according to age, thus suggesting that other differently regulated UGT isoforms were present and would account for the variations of 1-naphthol glucuronidation observed.

Cloning and initial characterization of human and mouse Spot 14 genes.

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.

LITTLE KEVIN: a program for the estimation of protein homology by analysing the amino acid compositions and sequences.

We present here a computational method based on the analysis of amino acid composition for performing comparisons between proteins. This user-friendly and reliable test is aimed at rapidly identifying, from data-base subsets, sequences--if necessary, partial sequences--which share similar amino acid compositions to the input composition (deduced from experimental results). Apparent molecular weight (as determined by SDS-PAGE) and artefactual modifications due to the experimental determination of the amino acid composition are taken into account to perform the comparison. This program thus constitutes a useful tool in searching for the probable identification of either non-sequenced proteins or peptides from hydrolysed proteins.