Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene

Azo pigments are used extensively as coloring agents in inks, paints and cosmetics. We have surveyed the literature for genotoxic and cancer data on nine colorants, which are structurally related to 1-phenylazo-2-hydroxynaphthalene (C.I. Solvent yellow 14). C.I. Solvent yellow 14 is metabolized by oxidative and peroxidative enzymes. Metabolically activated C.I. Solvent yellow 14 forms both RNA and DNA adducts. It induces liver nodules in rats upon oral administration. Although there is a mixture of negative and positive findings in short-term tests and in animal cancer studies, C.I. Solvent yellow 14 should be considered genotoxic. C.I. Pigment red 3 should be considered carcinogenic but is only weakly genotoxic. C.I. Solvent yellow 7, C.I. Pigment orange 5, C.I. Pigment red 4, and C.I. Pigment red 23 should be considered genotoxic. C.I. Pigment red 53:1 is not genotoxic, and observations of spleen tumors in male rats but not in female rats or mice seem to be related to toxic effects of high doses of C.I. Pigment red 53:1 in this organ. The data in the literature indicate that Pigment red 57:1 is not genotoxic or carcinogenic. We did not find sufficient data for a relevant evaluation of C.I. Pigment red 2 and C.I. Pigment red 64:1. Some of the colorants have in common the 2-amino-1-naphthol structure. This compound is not genotoxic. On the other hand, reductive cleavage of the azo bonds or hydrolysis of anilido bonds would produce aromatic amines, most of which have been under suspicion for genotoxicity or carcinogenicity. For C.I. Pigment red 53:1 and 57:1, sulphonated aromatic amines would be formed that are not genotoxic.     

Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.     

Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step

We have investigated the initial steps in the interaction between infectious salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line). Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on binding, uptake, and fusion of ISAV to SHK-1 cells and liposomes. As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto filters was neuraminidase sensitive. However, the increased binding induced by low pH was not neuraminidase sensitive, probably reflecting activation of a fusion peptide at low pH. By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculation with chloroquine or ammonium chloride, both electron and confocal microscopy showed accumulation of ISAV in endosomes and lysosomes. Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloroquine present during virus inoculation also caused a reduction in the synthesis of viral proteins in ISAV-infected cells as well as in the production of infective virus. These results indicate that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid environment of endosomes. This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classification of this virus as a member of the Orthomyxoviridae family.     

TNF-alpha, leptin, and lymphocyte function in human aging

Aging is associated with increased inflammatory activity and concomitant decreased T cell mediated immune responses. Leptin may provide a link between inflammation and T cell function in aging. The aim of the study was to investigate if plasma levels of tumor necrosis factor (TNF)-alpha were associated with leptin, circulating interleukin-2 receptors (sIL-2R), and phytohaemagglutinin (PHA) induced IL-2 production in whole blood in elderly humans. Circulating levels of TNF-alpha and sIL-2R were higher in elderly humans (N=42) compared to a young control group (N=37) whereas there was no difference with regard to IL-2 production. Furthermore, there were no age-related differences in serum levels of leptin. However, women had higher levels than men. In the elderly people, serum levels of leptin were correlated with TNF-alpha in univariate regression analysis and in a multiple linear regression analysis adjusting for the effect of gender and body mass index. Furthermore, TNF-alpha, but not leptin, was positively correlated to sIL-2R and negatively correlated to IL-2 production. In conclusion, increased plasma levels of TNF-alpha in aging is associated with poor IL-2 production ex vivo and lymphocyte activation in vivo. These associations do not seem to involve leptin.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Deconvolution can be used in electrooptic studies to correct for non-ideal electric excitation pulses only when the electric dipole moment of the studied molecules is predominantly induced

The electric field pulses used for most measurements of transient electrooptic properties such as birefringence and dichroism, are rectangular and assumed to be ideal, but in practice do all such pulses have non-zero rise and fall times. Claims have been made that this non-ideality may be taken into account by employing standard deconvolution techniques. We find that this approach yields exact results in the zero electric field limit when the electric dipole moment of the studied macromolecules is predominantly induced. However, for finite electric field strengths and/or macromolecules with partly or fully permanent electric dipole moments, we find that the deconvolution method yields erroneous estimates of the electrooptic relaxation times. When the decay time of the electric pulse and the electrooptic decay time are equal, and the system is operated in the Kerr domain, this systematic error for macromolecules with purely permanent electric dipole moment equals 37%. In a research field where the uncertainty of the reported relaxation times normally is assumed to be only a few percent this is an error that may seriously mislead unsuspecting users. We find that this systematic error can readily be avoided by employing standard numerical integration of a set of coupled first-order differential equations instead of the standard deconvolution techniques.     

Introducing selective supercritical fluid extraction as a new tool for determining sorption/desorption behavior and bioavailability of persistent organic pollutants in sediment

This review article intends to introduce the possibility of utilizing selective supercritical fluid extraction (SFE) as a tool to study sorption/desorption processes and bioavailability of persistent organic pollutants (POP) in sediment. Sorption/desorption behavior and bioavailability studies of POPs is a large research area, but still many unsolved problems exists. Therefore novel approaches to investigate mechanistic behavior of POPs in sediments are needed. Present literature on SFE points to the fact that selective SFE measurements can improve our knowledge, and recent investigations have been performed that demonstrate this. Results obtained with selective SFE can be connected to desorption of POPs in sediments under natural conditions in aquatic ecosytems. The ultimate goal is to use selective SFE as a way to determine the bioavailable fraction present within a matrix. A few preliminary results are presented here which may serve as a starting point for future studies.