Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Contrasting behavior of higher plant photosystem I and II antenna systems during acclimation

In this work we analyzed the photosynthetic apparatus in Arabidopsis thaliana plants acclimated to different light intensity and temperature conditions. Plants showed the ability to acclimate into different environments and avoid photoinhibition. When grown in high light, plants had a faster activation rate for energy dissipation (qE). This ability was correlated to higher accumulation levels of a specific photosystem II subunit, PsbS. The photosystem II antenna size was also regulated according to light exposure; smaller antenna size was observed in high light-acclimated plants with respect to low light plants. Different antenna polypeptides did not behave similarly, and Lhcb1, Lchb2, and Lhcb6 (CP24) are shown to undergo major levels of regulation, whereas Lhcb4 and Lhcb5 (CP29 and CP26) maintained their stoichiometry with respect to the reaction center in all growth conditions. The effect of acclimation on photosystem I antenna was different; in fact, the stoichiometry of any Lhca antenna proteins with respect to photosystem I core complex was not affected by growth conditions. Despite this stability in antenna stoichiometry, photosystem I light harvesting function was shown to be regulated through different mechanisms like the control of photosystem I to photosystem II ratio and the association or dissociation of Lhcb polypeptides to photosystem I.     

Phylogenetic relationships among the Lorisoidea as indicated by craniodental morphology and mitochondrial sequence data

The phylogeny of the Afro-Asian Lorisoidea is controversial. While postcranial data attest strongly to the monophyly of the Lorisidae, most molecular analyses portray them as paraphyletic and group the Galagidae alternately with the Asian or African lorisids. One of the problems that has bedevilled phylogenetic analysis of the group in the past is the limited number of taxa sampled for both ingroup families. We present the results of a series of phylogenetic analyses based on 635 base pairs (bp) from two mitochondrial genes (12S and 16S rRNA) with and without 36 craniodental characters, for 11 galagid and five lorisid taxa. The outgroup was the gray mouse lemur (Microcebus murinus). Analyses of the molecular data included maximum parsimony (MP), neighbor joining (NJ), maximum likelihood (ML), and Bayesian methods. The model-based analyses and the combined "molecules+morphology" analyses supported monophyly of the Lorisidae and Galagidae. The lorisids form two geographically defined clades. We find no support for the taxonomy of Galagidae as proposed recently by Groves [Primate Taxonomy, Washington, DC: Smithsonian Institution Press. 350 p, 2001]. The taxonomy of Nash et al. [International Journal of Primatology 10:57-80, 1989] is supported by the combined "molecules+morphology" analysis; however, the model-based analyses suggest that Galagoides may be an assemblage of species united by plesiomorphic craniodental characters.     

Iron deficiency differently affects metabolic responses in soybean roots

Iron deficiency responses were investigated in roots of soybean, a Strategy I plant species. Soybean responds to iron deficiency by decreasing growth, both at the root and shoot level. Chlorotic symptoms in younger leaves were evident after a few days of iron deficiency, with chlorophyll content being dramatically decreased. Moreover, several important differences were found as compared with other species belonging to the same Strategy I. The main differences are (i) a lower capacity to acidify the hydroponic culture medium, that was also reflected by a lower H(+)-ATPase activity as determined in a plasma membrane-enriched fraction isolated from the roots; (ii) a drastically reduced activity of the phosphoenolpyruvate carboxylase enzyme; (iii) a decrease in both cytosolic and vacuolar pHs; (iv) an increase in the vacuolar phosphate concentration, and (v) an increased exudation of organic carbon, particularly citrate, phenolics, and amino acids. Apparently, in soybean roots, some of the responses to iron deficiency, such as the acidification of the rhizosphere and other related processes, do not occur or occur only at a lower degree. These results suggest that the biochemical mechanisms induced by this nutritional disorder are differently regulated in this plant. A possible role of inorganic phosphate in the balance of intracellular pHs is also discussed.     

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.     

Vascular flows and transpiration affect peach (Prunus persica Batsch.) fruit daily growth

The relative contributions of xylem, phloem, and transpiration to fruit growth and the daily patterns of their flows have been determined in peach, during the two stages of rapid diameter increase, by precise and continuous monitoring of fruit diameter variations. Xylem, phloem, and transpiration contributions to growth were quantified by comparing the diurnal patterns of diameter change of fruits, which were then girdled and subsequently detached. Xylem supports peach growth by 70%, and phloem 30%, while transpiration accounts for approximately 60% of daily total inflows. These figures and their diurnal patterns were comparable among years, stages, and cultivars. Xylem was functional at both stage I and III, while fruit transpiration was high and strictly dependent on environmental conditions, causing periods of fruit shrinkage. Phloem imports were correlated to fruit shrinkage and appear to facilitate subsequent fruit enlargement. Peach displays a growth mechanism which can be explained on the basis of passive unloading of photoassimilates from the phloem. A pivotal role is played by the large amount of water flowing from the tree to the fruit and from the fruit to the atmosphere.     

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.     

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.     

To move or not to move? Semaphorin signalling in cell migration

Semaphorins were discovered 11 years ago as molecular cues for axon guidance that are conserved from invertebrates to humans. More than 20 semaphorin genes have been identified in mammals and their protein products are now known to be involved in a range of processes from the guidance of cell migration to the regulation of the immune response, angiogenesis and cancer. Plexins, either alone or in association with neuropilins, constitute high-affinity semaphorin receptors. However, other transmembrane molecules have been implicated in semaphorin receptor complexes, and interactions between plexins and a range of intracellular effectors have been reported. These data indicate that semaphorins might be able to elicit responses through more than one signalling pathway. Interestingly, according to recent findings, the semaphorin-dependent control of cell migration crucially involves integrin-based adhesive structures through which polarized cell-membrane protrusion is coupled to cytoskeletal dynamics. This review focuses on the mechanisms whereby semaphorins are thought to regulate cell migration.     

Vitamin E reduces amyloidosis and improves cognitive function in Tg2576 mice following repetitive concussive brain injury

Traumatic brain injury is a well-recognized environmental risk factor for developing Alzheimer's disease. Repetitive concussive brain injury (RCBI) exacerbates brain lipid peroxidation, accelerates amyloid (Abeta) formation and deposition, as well as cognitive impairments in Tg2576 mice. This study evaluated the effects of vitamin E on these four parameters in Tg2576 mice following RCBI. Eleven-month-old mice were randomized to receive either regular chow or chow-supplemented with vitamin E for 4 weeks, and subjected to RCBI (two injuries, 24 h apart) using a modified controlled cortical impact model of closed head injury. The same dietary regimens were maintained up to 8 weeks post-injury, when the animals were killed for biochemical and immunohistochemical analyses after behavioral evaluation. Vitamin E-treated animals showed a significant increase in brain vitamin E levels and a significant decrease in brain lipid peroxidation levels. After RBCI, compared with the group on regular chow, animals receiving vitamin E did not show the increase in Abeta peptides, and had a significant attenuation of learning deficits. This study suggests that the exacerbation of brain oxidative stress following RCBI plays a mechanistic role in accelerating Alphabeta accumulation and behavioral impairments in the Tg2576 mice.