Expression of AmphiNaC, a new member of the amiloride-sensitive sodium channel related to degenerins and epithelial sodium channels in amphioxus

Degenerins and amiloride-sensitive Na+ channels form a new family of cationic ion channels (DEG/NaC). DEG/NaC family emerged as common denominator within a metazoan mechanosensory apparatus. In this study, we characterized a new member of such family in amphioxus, Branchiostoma floridae. The AmphiNaC cDNA sequence encodes a protein showing amino acid residues characteristic of DEG/NaC family, such as two hydrophobic domains surrounding a large extracellular loop that includes cystein-rich domains; nevertheless its predicted sequence is quite divergent from other family members. AmphiNaC is expressed at early larval stage in some putative sensory epidermal cells in the middle of the body and in neurons of the posterior cerebral vesicle, as well as in some ventrolateral and mediolateral neurons of the neural tube. In late larvae, AmphiNaC expression is maintained in some neurons of the neural tube, and it is expressed in putative sensory epidermal cells of rostrum and mouth. The analysis of AmphiNaC gene expression pattern suggests that it might be involved in neurotransmission and sensory modulation.     

X-ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.     

Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P- and TCDD-spiked soils

Earthworms are useful indicators of soil quality and are widely used as model organisms in terrestrial ecotoxicology. The assessment of genotoxic effects caused by environmental pollutants is of great concern because of their relevance in carcinogenesis. In this work, the earthworm Eisenia andrei was exposed for 10 and 28 days to artificial standard soil contaminated with environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (1×10(-5), 1×10(-4), 2×10(-3)ppm). Micronucleus (MNi) induction was evaluated in earthworm coelomocytes after DNA staining with the fluorescent dye DAPI. In the same cells, the DNA damage was assessed by means of the alkaline comet assay. Induction of MNi in coelomocytes, identified according to standard criteria, was demonstrated. B[a]P exposure for 10 and 28 days induced a significant increase in MNi frequency. In TCDD-treated earthworms, a significant effect on chromosomal damage was observed at all the concentrations used; surprisingly, greater effects were induced in animals exposed to the lowest concentration (1×10(-5)ppm). The data of the comet assay revealed a significant increase in the level of DNA damage in coelomocytes of earthworms exposed for 10 and 28 days to the different concentrations of B[a]P and TCDD. The results show that the comet and MN assays were able to reveal genotoxic effects in earthworms exposed even to the lowest concentrations of both chemicals tested here. The combined application in E. andrei of the comet assay and the micronucleus test, which reflect different biological mechanisms, may be suggested to identify genotoxic effects induced in these invertebrates by environmental contaminants in terrestrial ecosystems.     

Modulating Memory Performance in Healthy Subjects with Transcranial Direct Current Stimulation Over the Right Dorsolateral Prefrontal Cortex

Objective

The role of the Dorsolateral Prefrontal Cortex (DLPFC) in recognition memory has been well documented in lesion, neuroimaging and repetitive Transcranial Magnetic Stimulation (rTMS) studies. The aim of the present study was to investigate the effects of transcranial Direct Current Stimulation (tDCS) over the left and the right DLPFC during the delay interval of a non-verbal recognition memory task.

Method

36 right-handed young healthy subjects participated in the study. The experimental task was an Italian version of Recognition Memory Test for unknown faces. Study included two experiments: in a first experiment, each subject underwent one session of sham tDCS and one session of left or right cathodal tDCS; in a second experiment each subject underwent one session of sham tDCS and one session of left or right anodal tDCS.

Results

Cathodal tDCS over the right DLPFC significantly improved non verbal recognition memory performance, while cathodal tDCS over the left DLPFC had no effect. Anodal tDCS of both the left and right DLPFC did not modify non verbal recognition memory performance.

Conclusion

Complementing the majority of previous studies, reporting long term memory facilitations following left prefrontal anodal tDCS, the present findings show that cathodal tDCS of the right DLPFC can also improve recognition memory in healthy subjects.     

Oxidative and reductive pathways of estrogens in hormone responsive and non-responsive human breast cancer cells in vitro

In order to measure the formation and degradation rates of estradiol by human breast cancer cells, after assessing the biochemical basis of hormone responsiveness and growth response to estrogens, we considered both responsive, estrogen receptor (ER) positive, and non-responsive, ER-negative, breast cancer cell lines, i.e. MCF7, ZR75-1 and MDA-MB231. To this end, we employed a novel “intact cell” approach which allows us, after 24 h incubation, to analyze several enzyme activities in sequence, concurrently with the monitoring of labeled precursor degradation. Our investigations led to the following evidence: (a) the reductive activity of the 17β-hydroxysteroid oxoreductase (17β-HSOR) appears to be higher than the oxidative only in responsive, ER-rich MCF7 and ZR75-1 cells, as also previously observed by others; (b) this activity is, on the contrary, much lower in MDA-MB231 cells and other unresponsive, ER-poor breast cancer cell lines; (c) conversely, the oxidative activity shows an opposite pattern, being limited in MCF7 and ZR75-1 cells and much higher in MDA-MB231 cells. Overall, a 17β-HSOR reductive pathway prevails in both MCF7 and ZR75-1 cells, whilst the oxidative pathway is prevalent in MDA-MB231 cells, leading to a large formation of estrone that is no further metabolized, at least in the experimental conditions used. Our results may provide a likely explanation of previous data on the different estrogen content of breast tumor tissues.     

Expression of collapsin response mediator proteins in the nervous system of embryonic zebrafish

Collapsin response mediator proteins (CRMPs also known as TUC, Drp, Ulip, TOAD-64) are cytosolic phosphoproteins that are involved in signal transduction during axon growth and in cytoskeletal dynamics. Here we report cloning and mRNA expression patterns of CRMP-1, -2, -3, -4 and, owing to a genome duplication in teleosts, two homologs of CRMP-5 (CRMP-5a and -5b) in embryonic zebrafish at 16 and 24 h post-fertilization (hpf). CRMPs are evolutionarily conserved and zebrafish CRMPs show amino acid identities of 76–90% with their homologs in humans, with the exception of CRMP-3, which shows only 67% homology. Between 16 and 24 hpf, expression of CRMPs generally increased in many regions of the CNS undergoing neuronal differentiation and axonogenesis, but not in the proliferative ventricular zone. Structures that were typically labeled by most, but not all the CRMP probes were the telencephalon, the nucleus of the tract of the post-optic commissure, the epiphysis, the nucleus of the medial longitudinal fascicle, clusters of hindbrain neurons, cranial ganglia, as well as Rohon-Beard neurons. No expression of CRMP mRNAs was observed outside the nervous system. Thus, expression patterns of different CRMP family members correlate with neuronal differentiation and axonogenesis in embryonic zebrafish.     

Catechol-o-methyltransferase and methoxyestradiols participate in the intraoviductal nongenomic pathway through which estradiol accelerates egg transport in cycling rats

Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E(2)-accelerated egg transport. Intrabursal application of OR486 prevented E(2) from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E(2) on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E(2) accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E(2) nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.     

Born among the ice: first morphological observations on two developmental stages of the Antarctic silverfish Pleuragramma antarcticum, a key species of the Southern Ocean

The Antarctic silverfish Pleuragramma antarcticum is a keystone species in the Southern Ocean ecosystem, providing one of the major links between lower and higher trophic levels. Despite the importance of this species, surprisingly little is known of its early development. The first spawning area for the silverfish has been recently identified in the near-shore of Terra Nova Bay (Ross Sea). Evidence indicates that spawning and embryo development occurs in the cryopelagic environment, below the seasonal pack-ice. In order to contribute to the knowledge of the life cycle of this very important Antarctic species, we carried out the first histological characterization on pre-hatching embryos and newly hatched larvae. Embryonated eggs and larvae of P. antarcticum were collected between late October and November 2005 at TNB through holes drilled into the sea ice. Embryonic stage just before hatching and the first post-hatching stage were the most abundant within our samples and thus were analysed using both macroscopic and histological approaches. Early life stages of the Antarctic silverfish revealed interesting features: the sensory system, foraging apparatus and heart appeared well developed, whereas the liver and gills were underdeveloped. Morphological details of the organogenesis were performed, providing the first substantial information on the development of P. antarcticum and representing a further steps towards the knowledge of the life cycle of this important Antarctic key species. An erratum to this article can be found at