Seasonal monitoring of blue mussel (Mytilus spp.) populations in a harbor area: A focus on responses to environmental factors and chronic contamination.

Coastal waters corresponding to macrotidal systems are among the most variable marine biotopes. Sessile animals as bivalve mollusks may however be found forming intertidal beds at high densities, as allowed by full adaptation to local conditions. A better knowledge of adaptive responses to environmental factors is required to foresee possible adverse effects of global change. At the sub-cellular level, transcriptional responses are among the earliest signals of environmental disturbances and they can reveal subtle and meaningful changes in organism exposed to stress. Three blue mussel (Mytilus spp.) populations inhabiting the Bay of Brest (France) in sites exposed to different levels of chronic pollution, from low to moderate, were surveyed upon a seasonal schedule, with special attention to the reproductive cycle. Major seawater parameters were monitored over a full-year in the framework of the S!RANO project, based on an automatic high frequency acquisition system installed aboard a ship of opportunity. The health status of mussels has been assessed by measuring a condition index and gametogenesis has been followed by histology. Selected biological responses to environmental stress were detected using a multimarker approach including expression of genes involved in chemical stress response and energetic metabolism, and cellular immune parameters. Environmental parameters showed deep seasonal variations which differed among sites. Most biological responses followed a seasonal pattern. Late winter and spring corresponded to an active reproduction period in the Bay of Brest. Earlier spawning was observed in harbor areas compared to the oceanic site and an altered physiological state was assumed in commercial harbor mussels during the reproductive period, suggesting that their health is compromised at this time of year. However, no signs of severe chemical stress were detected in both harbor mussel populations, which could reflect adaptive responses to adverse environmental conditions.

Attitude of medical students towards occupational safety and health: a multi-national study.

BACKGROUND: Work-related diseases contribute immensely to the global burden of diseases. Better understanding of attitudes of health care workers towards occupational safety and health (OSH) is important for planning. OBJECTIVE: To assess the attitude of medical students towards OSH around the globe. METHODS: A questionnaire assessing the attitude towards OSH was administered to medical and paramedical students of 21 Medical Universities across the globe. In the current study 1895 students, aged 18-36 years, from 17 countries were included. After having performed a principal components analysis, the associations of interest between the identified components and other socio demographic characteristics were assessed by multivariate linear regression. RESULTS: Principal component analysis revealed 3 components. Students from lower and lower-middle-income countries had a more positive attitude towards OSH, but the importance of OSH was still rated higher by students from upper-income countries. Although students from Asian and African continents showed high interest for OSH, European and South-Central American students comparatively rated importance of OSH to be higher. Paramedical students had more positive attitude towards OSH than medical students. CONCLUSION: The attitude of students from lower-income and lower-middle-income towards importance of OSH is negative. This attitude could be changed by recommending modifications to OSH courses that reflect the importance of OSH. Since paramedical students showed more interest in OSH than medical students, modifications in existing health care system with major role of paramedics in OSH service delivery is recommended.

A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs.

Development of an innovative and "green" stir bar sorptive extraction-thermal desorption-gas chromatography-tandem mass spectrometry method for quantification of polycyclic aromatic hydrocarbons in marine biota.

There is a growing awareness of the need to reduce the negative impact of chemical analyses on the environment and to develop new eco-friendly and sustainable analytical methods without compromising performance. In this study, we developed a "green" analytical method enabling the accurate and simultaneous routine analysis of 21 polycyclic aromatic hydrocarbons (PAHs) in reduced quantities (100mg and 1g wet weight (WW)) of marine biota samples (fish muscle, mussel and oyster tissues) using alkaline digestion combined with stir bar sorptive extraction-thermal desorption-gas chromatography-tandem mass spectrometry (SBSE-GC-MS/MS). The innovative method provides good selectivity and specificity for most compounds. In 1gWW samples, limits of quantification (LOQs) ranged from 1 to 10µg/kgWW in fish muscle and from 0.5 to 10µg/kgWW in mussel tissue. The method enables most analytes to be quantified below the restrictive limits established by the European Commission (2 and 10µg/kgWW in fish muscle and bivalve mollusc, respectively). Higher LOQs were obtained in 100mgWW samples ranging from 1 to 50µg/kgWW. Recovery and linearity were assessed for all analytes. The results were satisfactory for most compounds with recoveries ranging from 94% to 117% in 1gWW mussel samples at spike concentration of 10ng/gWW with standard deviation not exceeding 12%. However, results confirmed that the SBSE efficiency is affected by the complexity of biological matrices, especially for high molecular weight compounds in lipid-rich mussel tissue. Because of the matrix effects, matrix-matched calibrations were carried out. Validation was performed using the standard reference material 1974c with recovery ranging from 71% to 119% except for naphthalene, anthracene and benzo(e)pyrene that were therefore not validated. Overall, the developed method meets analytical validation criteria for most compounds. Thanks to the combination of alkaline digestion and SBSE, which greatly simplifies sample treatment and limits solvent use to ethanol, the developed method followed most green analytical chemistry principles.

Peroxiredoxin 6 gene: a new physiological and genetic indicator of multiple environmental stress response in Pacific oyster Crassostrea gigas.

Peroxiredoxin 6 is a 1-cysteine peroxiredoxin involved in antioxidant processes. We characterised the full-length cDNA and genomic sequence of the gene encoding peroxiredoxin 6 (CgPrx6) in the Pacific oyster, Crassostrea gigas. A phylogenetic analysis of Prx6 sequences showed that the CgPrx6 segregates between vertebrate and invertebrate groups. We analysed the expression of mRNA coding for CgPrx6 using RT-PCR in gills and digestive gland of oysters sampled in different contaminated and reference estuaries of the Atlantic French coast. We also studied CgPrx6 exon 6 polymorphism by PCR-SSCP in the same populations. Our results showed that CgPrx6 gene expression was highly regulated in the estuaries showing differential contamination levels, as expression increased with pollution level. Polymorphism analysis revealed no significant allelic frequency variations between populations. However, heterozygote deficiency seems to occur in the most contaminated estuaries, suggesting a potential selective effect of environmental stress on heterozygote frequency. Finally, the use of CgPrx6 as a possible marker to monitor stress exposure in disturbed ecosystems is discussed.

Description of endoscopic ventricular anatomy in myelomeningocele.

OBJECTIVE: The purpose of this work is to present our endoscopic neuroanatomical findings of a series of myelomeningocele and hydrocephalus patients, treated with endoscopic third ventricular cisternostomy (ETVC), in order to describe ventricular configuration abnormalities in this group of patients, in which this neurosurgical procedure has limited performance. METHOD: We checked the videos of 10 endoscopic third ventricular cisternostomies of myelomeningocele patients taken during 24 months as from December 1998. A previous guideline is designed to record anatomic variables in the lateral ventricles, IIIrd ventricle, and basal cisterns. The topic is analyzed in view of the necropsy and imaging background data. RESULTS: The ETVC of lateral ventricles showed: absence of septum (9/10); absence of anteroseptal vein (8/10); absence of choroid plexus and thalamostriate vein (0/10); absence of fornix (1/10): small foramen of Monro (4/10). The ETVC of the IIIrd ventricle showed: impossibility of recognizing any mammillary bodies (4/10); presence of septations (5/10); presence of atypical veins in the floor (6/10); translucent floor (5/10); floor umbilications (5/10); absence of infundibulum (4/10); arachnoid adherences (7/10); and visual contact of basilar artery (4/10). CONCLUSION: There are categorical structural alterations in the ventricular system of myelomeningocele patients that are well correlated with previous necropsy and imaging reports. The ventricular system of dysraphic children presents severe anatomic alterations, which alter the reference points of the classical endoscopic third ventricular cisternostomy.

Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas.

This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.

Genetic responses to metal contamination in two clams: Ruditapes decussatus and Ruditapes philippinarum.

Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.

Cloning of a metallothionein gene and characterization of two other cDNA sequences in the Pacific oyster Crassostrea gigas (CgMT1).

Metallothionein (MT) genes encode essential metal-binding proteins involved in metallic homeostasis and detoxification in living organisms. Here, we describe the structure of the first Pacific oyster Crassostrea gigas metallothionein (CgMT1) gene and the sequences of two other MT cDNA. The CgMT1 gene sequence contains three coding exons plus a 5' entirely non-coding exon, and the predicted protein contains 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The three cDNA sequences present few substitutions in either coding sequence or UTRs. Induction of these MT-mRNA in heavy metal-treated oysters (i.e. cadmium) was confirmed by Northern blot analysis and RT-PCR and suggests a potential specific tissue expression rate. Southern blot analysis suggested the presence of multiple CgMT genes, and allowed the detection of restriction fragment length polymorphisms (RFLPs). Although the CgMT1 coding sequence showed 30-73% nucleotide identities with known sequences in other mollusks, it included the specific motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X-X-X-Cys-X-Cys-X(5)-Cys-X-Cys-Lys found in Mollusk family 2. Marine bivalves are commonly used as pollution bioindicators, thus the development of genetic markers based on CgMT1 polymorphism will allow a monitoring of heavy metal exposure in anthropogenically disturbed ecosystems.

Cloning and characterization of a gene coding for a novel metallothionein in the Pacific oyster Crassostrea gigas (CgMT2): a case of adaptive response to metal-induced stress?

Cases of heavy metal resistance acquisition have already been demonstrated in eukaryotes, which involve metallothionein (MT) gene duplication or amplification mechanisms. We characterized in a marine bivalve, Crassostrea gigas, a gene coding for an unusual MT, which has never been described in other species. Our results illustrate a unique case of exon duplication and rearrangement in the MT gene family. The particular organization of the third exon of this gene allows the synthesis of a MT that presents a higher metal ion binding capacity compared to previously described MTs. The formation of a supplementary third structural beta-domain is proposed to explain results obtained in in vitro experiments. Differences in the metal responsive element (MRE) copy number and MRE core sequence observed in the promoter of CgMT2 also suggest differential regulation of CgMT2 transcription and possible implication in the detoxification processes.

A tau fragment containing a repetitive sequence induces bundling of actin filaments.

Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on tau with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of tau protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the tau peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the tau peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6-4.7 resulted in a higher effect of tau peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for tau protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of epsilon-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a tau repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on tau for the dynamics of the interconnections between actin filaments and microtubules.

High-level expression of human asparagine synthetase and production of monoclonal antibodies for enzyme purification.

In order to obtain large quantities of extremely pure human asparagine synthetase for detailed kinetic and structural studies, its gene was cloned into a 2mu plasmid (pBS24.1GAS) suitable for replication in a Saccharomyces cerevisiae cir0 strain (AB116). In this construct, the transcription of the asparagine synthetase gene is regulated by the alcohol dehydrogenase II/glyceraldehyde-3-phosphate dehydrogenase promoter, which is subject to glucose repression. The expression of the enzyme was allowed to take place in yeast minimal medium containing D-galactose as the only sugar nutrient. Eleven monoclonal antibodies to recombinant human asparagine synthetase were produced and one of them was selected to make immunoaffinity resins. After single-step immunoaffinity chromatography, more than 1.2 mg of homogeneous enzyme was obtained from the total cell extract from a 100-ml yeast culture. The yield of pure enzyme was over 100-fold higher than that of a previously reported yeast expression system. SDS-PAGE analysis showed the enzyme to be extremely pure and isoelectric focusing gel electrophoresis showed that the enzyme has an isoelectric point of 7.5. Immunoaffinity-purified recombinant human asparagine synthetase demonstrated both glutamine-dependent and ammonia-dependent asparagine synthetase activities, as well as glutaminase activity.

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.

Antigen shedding from the surface of the infective stage larvae of Dirofilaria immitis.

A qualitative and quantitative analysis was made of the release of surface-associated molecules from developing Dirofilaria immitis infective-stage larvae (L3). D. immitis L3s were labelled with 125I using an Iodogen catalysed reaction and either maintained in culture or placed in chambers that were implanted into Lewis rats. The larvae released 10-20% of the labelled material each day during the first 4 days of in vitro and in vivo development. The loss of surface-labelled peptides from developing larvae corresponded with an increase in the amount of trichloroacetic acid-precipitable radioactivity found in the culture medium. SDS-PAGE analysis of the labelled material showed that the same 35 and 6 kDa components found in larval extracts were shed into culture medium by the developing parasites. Metabolic labelling studies and experiments in which larvae were labelled after different times in culture indicated that, once released, the surface-associated molecules were not replaced, and that this net loss of surface peptides resulted in a reduction in the antigenic potential of the cuticular surface. Antibodies from both immunized rabbits and naturally infected dogs immunoprecipitated the 35 kDa component. In contrast, the 6 kDa molecule was not recognized by the antibodies in any of the sera tested. Shedding of surface peptides and reducing surface antigenicity may represent mechanisms by which D. immitis infective-stage larvae evade immune attack.

Dirofilaria immitis: studies on anti-microfilarial immunity in Lewis rats.

The effect of vaccination on rates of microfilarial clearance using Dirofilaria immitis in male Lewis rats was examined. Animals were immunized with whole, dead microfilariae or a PBS extract of microfilariae in Freund's adjuvant. The immunized animals, as well as untreated and adjuvant controls, were challenged intravenously with 4 x 10(5) viable microfilariae (mf). The duration of microfilaremia was 15.5 days in rats vaccinated with whole mf, 17.7 days in those vaccinated with a PBS extract, 36.3 days for those vaccinated with adjuvant alone, and greater than 70 days for the untreated group. Analysis of the anti-microfilarial IgG response by ELISA and Western blots demonstrated that immunization induced significant amounts of antibody against high molecular weight peptides, particularly a peptide located at 105 kDa. Antibody levels in both groups of immunized animals continued to rise following challenge, reaching peak levels of 78-80 micrograms/ml on the day of microfilarial clearance. Decreasing microfilaremia following challenge was associated with an enhanced recognition of low molecular weight peptides.

Dirofilaria immitis: biochemical and immunological characterization of the surface antigens from adult parasites.

The molecules associated with the surface of adult Dirofilaria immitis were identified and characterized employing IODO-GEN-mediated surface labeling methods. D. immitis female and male parasites were found to have a limited number of surface-associated proteins (17.5, 16, and 14.5 kDa) and glycoproteins (49 and 20 kDa) which were readily extracted from parasite homogenates in the absence of detergent. The major surface labeled proteins and glycoproteins were antigenic in rabbits, but appeared to elicit only a weak humoral response in dogs with patent dirofilariasis. In addition, a 10- to 6-kDa surface-associated glycolipid was identified which may form a coat on the outside of the parasite and play a role in immune evasion. In immunoprecipitation experiments, the glycolipid was not recognized by the antibodies from rabbits exposed to the glycolipid or by antibodies in the sera of patently infected animals. The glycolipid and the 14.5-kDa surface protein were selectively released by the adult parasite during in vitro culture.

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment.

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.

Comparison of glycine metabolism in mouse lymphoma cells either sensitive or resistant to L-asparaginase.

Previous work suggested a relationship between glycine metabolism and the effect of L-asparaginase upon tumor cells. Therefore, L5178Y (sensitive) or L5178Y/L-ASE (resistant) ascites lymphoma cells were incubated with 14C-labeled glyoxylate, glycine, serine, or asparagine, and the metabolism to other amino acids was measured by high performance liquid chromatography. Metabolic differences between the two cells lines were found. Under control conditions, the interconversion rate of glycine and serine via serine hydroxymethyltransferase (SHMT) was higher in sensitive than in resistant cells. The transformation rate of glyoxylate to serine was also higher in sensitive cells. These results may indicate a difference in the activity of SHMT. An alternate explanation would be that transport or diffusion of serine and glycine into sensitive cells is greater than into resistant cells. Several crucial metabolic differences were observed between the two cell types when L-asparaginase was added. A key difference is the decrease of glycine synthesis from glyoxylate observed in the sensitive cells compared to resistant cells which show no change. This suggests that asparagine is used for transamination of glyoxylate. Also, only sensitive cells appear to compensate for L-asparaginase-induced loss of glycine formation from glyoxylate by increasing glycine synthesis from serine. Alterations in sensitive tumor glycine metabolism may be an important function of L-asparaginase anticancer activity.

Dose rate dependence of radiation induced IgG membrane receptor alteration [1].

Mouse lymph node lymphocytes are irradiated at different dose rates and the B-cell receptors to anti IgG are tested. The expression of receptors is inhibited by irradiation. It is shown that the effectivity of irradiation increases with decreasing dose rate suggesting that membrane damage may be important for situations of chronic irradiation or implant radiotherapy.