The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 microm. Considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitrite-mediated damage to biomolecules and to cultured human SH-SY5Y cells. H2S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 microm). H2S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S.?flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S.?flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S.?flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S.?flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect-plant interactions.     

SEXUAL SELECTION IN AMERICAN TOADS: A TEST OF A GOOD-GENES HYPOTHESIS

Adaptive mate choice in species lacking male resource control and/or paternal care might be maintained by selection because preferred males sire genetically superior offspring. For such a process to occur, some male phenotypic trait(s) must both reliably indicate male genetic quality and influence the pattern of mate choice by females. In American toads, Bufo americanus, male body length has been documented to influence female mating patterns: females usually mate with males that are larger than average. However, the relationship between male size and male genetic quality is unknown. We conducted a controlled breeding experiment using 48 sires and 19 dams to determine if larger males sire offspring with superior larval performance characteristics (greater survival to metamorphosis, larger mass at metamorphosis, and earlier metamorphosis). We also aged each sire to test the hypothesis that older males are, on average, genetically superior to younger males. We crossed each female with three sires representing three body size categories (mean and 1 SD ± mean snout-ischium length). Hatchlings (500 from each cross) were reared to metamorphosis in seminatural ponds in the field. Metamorph weight (log transformed) and age at metamorphosis showed significant heritability and were genetically correlated with each other. Hence, sires differed in genetic quality. However, none of the three measures of offspring performance was correlated with sire body size or age. Thus, we obtained no support for the prediction that sire body size or age is related to genetic quality.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Microfilariae in Galápagos penguins (Spheniscus mendiculus) and flightless cormorants (Phalacrocorax harrisi): genetics, morphology, and prevalence

Galapagos penguins (Spheniscus mendiculus) and flightless cormorants (Phalacrocorax harrisi) live in small, isolated populations on the westernmost islands of Isabela and Fernandina in the Galápagos Islands, Ecuador. Between August 2003 and February 2005, 4 field trips, 2 in the cool, dry season (August 2003 and August 2004) and 2 in the hot, rainy season (March 2004 and February 2005), were undertaken; 298 Galápagos penguins and 380 cormorants were sampled for prevalence and intensity of hemoparasites. Microfilariae were found in both the penguins and the cormorants. Blood smears were negative for the presence of other species of hemoparasites. Overall prevalence of microfilariae across seasons was 42.0% in cormorants and 13.8% in the penguins. Intensity of infection was generally low (mean = 3.2-31.7 in 25 fields across seasons and species) with the exception of a few individuals with markedly high intensities of parasites (>300 in 25 fields in 1 cormorant). Prevalence of microfilariae increased significantly over the 4 sampling periods for cormorants, but not for penguins. Prevalences were significantly higher in cormorants than in penguins for 3 of the 4 collecting trips. Male penguins had higher prevalences than females; however, there were no gender differences in cormorants. No relation was detected between body mass and either presence or intensity of parasitism. Morphological characteristics of the microfilariae are also described and specimens from each host species were similar in all characters measured. DNA sequence data from the mitochondrial cytochrome c oxidase subunit I gene were consistent with the morphological evidence and together demonstrate that the penguins and cormorants are likely to be infected with the same species of microfilariae.