Process type is the key driver of the fate of organic micropollutants during industrial scale treatment of organic wastes.

Organic micropollutants (OMPs) such as polycyclic aromatic hydrocarbons, nonylphenols and pharmaceutical products are ubiquitous in organic wastes generated by most human activities. Those wastes are mainly recycled by land spreading, most often after treatments, such as liming, dewatering, composting or anaerobic digestion. It has been shown essentially at lab scales that biological treatments have an effect on the removal of some OMPs. However, less is known on the role of each step of industrial treatment lines combining physico-chemical and biological treatments on the OMP fate and removal. The present study focuses on the impact of waste treatment on the fate of 53 OMPs along 10 industrial treatment lines treating urban, agricultural wastes or mixtures. The combination of studying a diversity of organic wastes and of OMPs with different characteristics (solubility, ionic charges, hydrophobicity etc.), sampling in situ industrial sites, quantifying native OMP concentrations and looking at each step of complete treatment lines allows for a global and representative view of the OMP fate in the French organic waste treatment sector. Less studied wastes, i.e. territorial mixtures, revealed intermediate OMP contents and compositions, between urban and agricultural wastes. Dewatering and liming, usually dismissed, had a noticeable effect on concentrations. Anaerobic digestion and composting had significant effects on the removal of all pollutant families. Combination of processes enhanced most OMP dissipation. Here we showed for the first time that the process type rather than the waste origin affects dissipation of organic micropollutants. Such data could be used to build and validate dynamic models for the fate of OMPs on solid waste treatment plants.

The adaptive metabolic response involves specific protein glutathionylation during the filamentation process in the pathogen Candida albicans.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685). We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen.

Kinetic analyses guide the therapeutic decision in a novel form of moderate aromatic Acid decarboxylase deficiency.

BACKGROUND: Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder resulting in a combined dopamine and serotonin deficiency. About 50% of the cases set in the neonatal period. Here, we report an atypical clinical presentation with moderate symptoms. PATIENT: At 10months old, the patient presented paroxysmal eye movements without seizures, and feeding difficulties which were attributed to gastroesophageal reflux. She was investigated at the age of 7years, because of orofacial dyspraxia, hypomimie, axial hypotonia and focal segmental dystonia, bilateral ptosis, without evidence for cognitive impairment. RESULTS: HVA [110nM; (reference value (rv): 202-596)] and HIAA (12nM; rv: 87-366) decreased, OMD (520nM; rv: 5-60) and 5-HTP (56nM; rv: 2-16) increased in CSF. We confirmed the diagnosis of AADC deficiency because the activity in plasma was low: 4pmol/min/ml; rv: 16-137. The kinetic analysis revealed a sixfold increase in the apparent affinity for L-dopa (4.26mM; control=0.71), but the V (max) was unchanged (37.5pmol dopamine/min/ml; control=39.1), suggesting a modification in the substrate binding-site. Molecular analysis revealed two heterozygous mutations in the DDC gene: c1040G > A; pR347Q already described, and a novel mutation c478C > T, pR160W. CONCLUSION: (1) CSF neurotransmitters metabolites suggested a moderate AADC deficiency; (2) The initial velocity saturation curve for L-dopa displayed a cooperative ligand binding behavior, in keeping with the modifications of the three-dimensional structure, induced by the amino acid substitutions (3) The treatment combination of L-dopa with pyridoxine dramatically improved the quality of life, the fatigability, and the paroxysmal eye movements.

The severity of phenotype linked to SUCLG1 mutations could be correlated with residual amount of SUCLG1 protein.

BACKGROUND: Succinate-CoA ligase deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Mutations in SUCLA2, the gene encoding a ß subunit of succinate-CoA ligase, have been reported in 17 patients until now. Mutations in SUCLG1, encoding the α subunit of the enzyme, have been described in two pedigrees only. METHODS AND FINDINGS: In this study, two unrelated patients harbouring three novel pathogenic mutations in SUCLG1 were reported. The first patient had a severe disease at birth. He was compound heterozygous for a missense mutation (p.Pro170Arg) and a c.97+3G>C mutation, which leads to the complete skipping of exon 1 in a minigene expression system. The involvement of SUCLG1 was confirmed by western blot analysis, which showed absence of SUCLG1 protein in fibroblasts. The second patient has a milder phenotype, similar to that of patients with SUCLA2 mutations, and is still alive at 12 years of age. Western blot analysis showed some residual SUCLG1 protein in patient's fibroblasts. CONCLUSIONS: Our results suggest that SUCLG1 mutations that lead to complete absence of SUCLG1 protein are responsible for a very severe disorder with antenatal manifestations, whereas a SUCLA2-like phenotype is found in patients with residual SUCLG1 protein. Furthermore, it is shown that in the absence of SUCLG1 protein, no SUCLA2 protein is found in fibroblasts by western blot analysis. This result is consistent with a degradation of SUCLA2 when its heterodimer partner, SUCLG1, is absent.

The first founder DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome.

Deoxyguanosine kinase (dGK) deficiency is a frequent cause of mitochondrial DNA depletion associated with a hepatocerebral phenotype. In this study, we describe a new splice site mutation in the DGUOK gene and the clinical, radiologic, and genetic features of these DGUOK patients. This new DGUOK homozygous mutation (c.444-62C>A) was identified in three patients from two North-African consanguineous families with combined respiratory chain deficiencies and mitochondrial DNA depletion in the liver. Brain MRIs are normal in DGUOK patients in the literature. Interestingly, we found subtentorial abnormal myelination and moderate hyperintensity in the bilateral pallidi in our patients. This new mutation creates a cryptic splice site in intron 3 (in position -62) and is predicted to result in a larger protein with an in-frame insertion of 20 amino acids. In silico analysis of the putative impact of the insertion shows serious clashes in protein conformation: this insertion disrupts the alpha5 helix of the dGK kinase domain, rendering the protein unable to bind purine deoxyribonucleosides. In addition, a common haplotype that segregated with the disease in both families was detected by haplotype reconstruction with 10 markers (microsatellites and SNPs), which span 4.6 Mb of DNA covering the DGUOK locus. In conclusion, we report a new DGUOK splice site mutation that provide insight into a critical protein domain (dGK kinase domain) and the first founder mutation in a North-African population.

Cooperativity and high pressure: stabilization of the R conformation of the allosteric aspartate transcarbamylase under the influence of pressure.

The allosteric enzyme aspartate transcarbamylase (ATCase) from E. coli shows homotropic cooperative interactions between its six catalytic sites for the binding of the substrate aspartate. This cooperativity is explained by the transition of the enzyme from a conformation which has a low affinity for aspartate (T state) to a conformation with high affinity (R state). The crystallographic structures of these two conformations are known to a resolution of 2.5 A and 2.1 A, respectively, and they reveal an important difference in the quaternary structure of the protein. Enzyme kinetics under high pressure were used to study the transition between the two states. It appears that in the presence of a low concentration of aspartate, conditions under which the enzyme is essentially in the T state, pressure promotes the transition to the R state, the maximal effect being observed at 120 MPa. This transition is accompagnied by a significant deltaV. This observation is in accordance with the change in the protein surface exposed to the solvent, and with the increased number of water molecules bound to the protein. Since the partial specific volume of the enzyme does not change significantly during the T to R transition, the negative deltaV is only related to the change in hydration of the protein. This result emphasizes a significant role of the protein-solvent interactions in this important regulatory conformational change.

Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

Remarkable changes occur during aging in the testis and epididymis of the Brown Norway rat. A dramatic increase in the number of halo cells, which are present in the epididymal epithelium and originate from the immune system, is found in animals of increasing age. Halo cells have been postulated to be either lymphocytes or monocytes. We hypothesized that halo cells are a mixture of different immune cells and that their relative composition changes with age. To verify this hypothesis, markers for helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, and monocytes-macrophages were used to identify the major categories of immune cells in the epididymides of Brown Norway rats ranging in age from 3 to 24 mo. The numbers of immunocompetent cells in the epididymis were determined in relation to age, epididymal segment, and luminal content. We found that monocytes, helper T lymphocytes, and cytotoxic T lymphocytes belong to the population of halo cells. In addition, a segment-specific increase with age in the number of these immune cells was noted. Finally, we report a segment-specific recruitment of cytotoxic T lymphocytes and monocytes-macrophages in the epididymal epithelium of aged rats whose epididymal lumen contained few spermatozoa. We postulate that accumulation of damaged epithelial cells and antigens of germ cell origin, leaking through a dysfunctional blood-epididymis barrier, may contribute to the active recruitment of immune cells with age.

Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain.

The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site.

The effects of aging on the seminiferous epithelium and the blood-testis barrier of the Brown Norway rat.

Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.

Loss of alpha 1 connexin does not alter the prenatal differentiation of pancreatic beta cells and leads to the identification of another islet cell connexin.

Connexin alpha 1, also referred to as Cx43, has thus far been the only gap junction protein identified between the hormone-producing cells of pancreatic islets. To investigate whether loss of this connexin affects the development of endocrine pancreas and the differentiation of insulin-producing beta cells, we have taken advantage of a transgenic line in which the gene coding for connexin alpha 1 had been functionally deleted by homologous recombination. Analysis of pancreas at embryonal day 19.5 (E 19.5) after immunostaining for the four main types of islet hormones, showed that islet cell development was similar in homozygous transgenic mice that completely lacked alpha 1 connexin, in mice that were heterozygous for the transgene, and in age-matched controls with a genetic background similar to that of the transgenic animals. In particular, the three animal groups featured beta cells that had a similar insulin content and ultrastructural organization, including the presence of typical gap junction plaques on the membrane. However, quantitative analysis of freeze-fractured membranes showed that these plaques were less frequent in the transgenic mice lacking alpha 1 connexin. This finding prompted us to revisit the connexin pattern of normal pancreatic beta cells. Using RT-PCR amplification and primers specific for nine of the mammalian connexins, we have found that normal rat and mouse pancreas contain six connexin transcripts, including one that codes for alpha 6 connexin, a protein also referred to as Cx45. This transcript was also identified in isolated pancreatic islets, in FACS-purified suspensions of primary beta cells and in the insulin-producing cells of an experimental tumor. Using antibodies, we found that connexin alpha 6 is expressed by the latter cells, as well as by pancreatic fibroblasts and epithelial duct cells. The data show that pancreatic islets have a normal prenatal development in mice that no longer express alpha 1 connexin. They further provide evidence that normal and tumoral insulin-producing cells natively coexpress connexins alpha 1 and alpha 6.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

Paternal age affects fertility and progeny outcome in the Brown Norway rat.

OBJECTIVE: To investigate the effects of paternal age on fertility and progeny outcome using the Brown Norway rat model. DESIGN: Controlled prospective study. SETTING: McIntyre Animal Centre, McGill University, Montreal, Quebec, Canada. INTERVENTION(S): Brown Norway male rats of increasing age were mated to young Sprague-Dawley females. MAIN OUTCOME MEASURE(S): Pregnancy outcome was assessed by counting the numbers of corpora lutea, resorptions, and live fetuses on day 20 of gestation. To evaluate progeny outcome, pups were examined for external malformations and weighed daily for 2 months. RESULT(S): There were no significant changes in the numbers of resorptions, offspring, or in the incidence of external malformations. However, there was an increase in preimplantation loss (corpora lutea minus implantation sites) in litters fathered by older males. Furthermore, a significant decrease in the average fetal weight was found with increasing paternal age. A significant increase in neonatal deaths for progeny fathered by older males also was found. CONCLUSION(S): These results indicate that the quality of spermatozoa decreases as males age.

Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins.

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.

Segment-specific morphological changes in aging Brown Norway rat epididymis.

In aging Brown Norway rats, both spermatogenesis and steroidogenesis decrease. Little is known about changes in the epididymis during aging. However, since the two major components entering the epididymis from the testis change, we hypothesized that epididymal histology would be affected by advancing age. The epididymides of Brown Norway rats ranging in age from 3 to 24 mo were prepared for light and electron microscopy. Striking quantitative and qualitative changes were noted. There was an age-dependent increase in the thickness of the basal membrane and in the number of halo cells. There were also major segment-specific changes in the appearance of cells along the epididymis with age. At 12 mo, basal cells in the initial segment emitted pseudopods into the basement membrane. By 18 mo, in the caput epididymidis, clear cells were filled with lysosomes; these cells frequently showed bulging protrusions into the lumen. In the corpus epididymidis, the cytoplasm of principal cells had numerous large lysosomes both below and above the nucleus; apical cells were usually occupied by one giant membranous lysosome. In the proximal cauda, clear cells became filled with dense lysosomes, and principal cells presented large clear vacuoles; debris from spermatozoa was found in the larger vacuoles. In summary, aging of the epididymis was accompanied by the emergence of characteristic features of aging and activation of the immune system. Furthermore, there were many cell- and segment-specific changes. Finally, these changes were not related to the presence of spermatozoa, often preceding their disappearance, thus indicating that there may be an intrinsic mechanism of aging in epididymal epithelial cells.

Effects of cysteine modification on the activity of the cGMP-gated channel from retinal rods.

The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide ("activation") at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments ("inhibition"). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.

Allosteric regulation of carbamoylphosphate synthetase-aspartate transcarbamylase multifunctional protein of Saccharomyces cerevisiae: selection, mapping and identification of missense mutations define three regions involved in feedback inhibition by UTP.

The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase). The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb). All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain. In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms. Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain. They are clustered in two regions of this domain which must be directly involved in the feedback process.

Lymphoproliferative response to mitogen (ConA) after treatment of rats with gentamicin and doxycyclin: relevance to human therapeutics.

Interaction of xenobiotics with the immune system may result in undesirable effects such as immunosuppression. In the future, it is probable that immunological screening tests may be required for safety evaluation of new drug candidates as an adjunct to more traditional toxicity testing. The lymphoproliferative response to mitogens has been proposed as a screening test. This test has been set up and validated in mice, but will probably have to be done in rats, the species of choice for drug safety studies. This test has been adapted to rats, using both in vivo and in vitro treatments, and used to validate drugs, including doxycyclin and gentamicin (with cyclosporin A and hydroxycortisone as controls) at the dose levels used in toxicology studies. Discrepant results obtained with doxycyclin and hydroxycortisone (effect in vitro but not in vivo) suggest that further validation studies are necessary in order to assess the reliability of this test.