Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response.     

Zebrafish and medaka as models for bone research including implications regarding space-related issues

Summary. Teleost fish develop bones directly from mesenchymal condensations and from cartilage precursors. At the cellular level, the involved cell populations share many features with their mammalian counterparts. In addition, several genes are already described in fish showing high homology in amino acid sequence and expression with the corresponding genes of tetrapods that are involved in bone metabolism. Therefore, analysis of the underlying molecular mechanism in fish, in particular zebrafish and medaka, will increase the knowledge in teleosts. Furthermore, it will help to identify novel genes and regulatory pathways of bone homeostasis and skeletal disorders also in higher vertebrates, including disorders caused by altered gravity. Correspondence and reprints (present address): Department of Physiological Chemistry I, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Federal Republic of Germany.     

Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes

Protein tyrosine nitration may be relevant for the pathogenesis of hepatic encephalopathy (HE). Infections, sepsis, and trauma precipitate HE episodes. Recently, serum levels of tumor necrosis factor (TNF)-alpha were shown to correlate with severity of HE in chronic liver failure. Here the effects of inflammatory cytokines on protein tyrosine nitration in cultured rat astrocytes and rat brain in vivo were studied. In cultured rat astrocytes TNF-alpha (50 pg/ml-10 ng/ml) within 6h increased protein tyrosine nitration. TNF-alpha-induced tyrosine nitration was related to an increased formation of reactive oxygen and nitrogen intermediates, which was downstream from a NMDA-receptor-dependent increase of intracellular [Ca(2+)](i) and nNOS-catalyzed NO production. Astroglial tyrosine nitration was also elevated in brains of rats receiving a non-lethal injection of lipopolysaccharide, as indicated by colocalization of nitrotyrosine immunoreactivity with glial fibrillary acidic protein and glutamine synthetase, and by identification of the glutamine synthetase among the tyrosine-nitrated proteins. It is concluded that reactive oxygen and nitrogen intermediates as well as protein tyrosine nitration by inflammatory cytokines may alter astrocyte function in an NMDA-receptor-, Ca(2+)-, and NOS-dependent fashion. This may be relevant for the pathogenesis of HE and other conditions involving cytokine exposure the brain.     

Haematology of swordtail, Xiphophorus helleri. II: cellular composition and isolation of blood cells from kidney of Xiphophorus helleri

The quantitative determination of blood cells in the kidney of swordtail, Xiphophorus helleri , was determined by light microscopical observations. Lymphocytes/thrombocytes and neutro-philic granulocytes (32.4 and 31.6%, respectively) represent the main blood cells in kidney. High amounts of blastic cells in different mitotic phases indicate the role of the kidney as a major haematopoietic organ in this fish species. Additionally, a good and reproducible method for the isolation of accessory and immunoreactive cells was developed using density gradient centrifugation.     

Differential effects of testosterone,dihydrotestosterone and estradiol on carotenoid deposition in an avian sexually selected signal

Recent studies have demonstrated that carotenoid-based traits are under the control of testosterone (T) by up-regulation of carotenoid carriers (lipoproteins) and/or tissue-specific uptake of carotenoids. T can be converted to dihydrotestosterone (DHT) and estradiol (E2), and variation in conversion rate may partly explain some contradictory findings in the literature. Moreover, most studies on the effect of T on sexual signals have focused on the male sex only, while in many species females show the same signal, albeit to a lesser extent. We studied the effects of T, DHT, and E2 treatment in male and female diamond doves Geopelia cuneata in which both sexes have an enlarged red eye ring, which is more pronounced in males. We first showed that this periorbital ring contains very high concentration of carotenoids, of which most are lutein esters. Both T and DHT were effective in enhancing hue, UV-chroma and size in both sexes, while E2 was ineffective. However, E2 dramatically increased the concentration of circulating lipoproteins. We conclude that in both sexes both color and size of the secondary sexual trait are androgen dependent. The action of androgens is independent of lipoproteins regulation. Potential mechanisms and their consequences for trade-off are discussed.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing