Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2. To identify genes involved in neuronal regeneration we performed three GEM experiments, which included changes in gene expression in rat dorsal root ganglia during the healing of experimentally injured sciatic nerve, in regenerating neonatal opossum spinal cord, and during lipopolysaccharide stimulation of primary cultures of rat Schwann cells. Finally we have monitored genes involved in the recovery phase of the inflammatory disease of the rat spinal cord, experimental allergic encephalomyelitis, as well as those responsible for protection from oxidative stress in a glutamate-resistant rat hippocampal cell line. Analysis of the results of the approximately 70,000 data points collected is presented.     

From the Hiatus Model to the Diffuse Discontinuities: A Turning Point in Human-Animal Studies

In twentieth-century continental philosophy, German philosophical anthropology (Max Scheler, Helmuth Plessner, and Arnold Gehlen) can be seen as a sort of conceptual laboratory devoted to human/animal research, and, in particular, to the discontinuity between human and non-human animals. Its main notion—the idea of the special position of humans in nature—is one of the first philosophical attempts to think of the specificity of humans as a natural and qualitative difference from non-human animals. This school of thought correctly rejects both the metaphysical and/or religious characterisations of humans, and the positivistic gradualism, that sees the human being as an animal endowed with a greater degree of certain faculties (intelligence, etc.). At the same time, German philosophical anthropology still takes it for granted that such natural-qualitative novelty is unique in the realm of the living, that in correspondence with humans there is the hiatus, the discontinuity par excellence. The semiotic side of this view is the distinction between signs and symbols developed by Ernst Cassirer and Susanne Langer: animal signs would be mere proxies for perceptive elements or stimuli, whereas only human symbols could convey complex representation of objects and situations. The goal of this contribution is to criticise the alleged uniqueness of the hiatus and its semiotic implications through the opposite approach of the diffuse discontinuities. This approach that focuses on the semiotic traits of different species-specific environments (Umwelten) can be traced back to Jakob von Uexküll’s biosemiotical phenomenology, which thinks of discontinuities as a normal phenomenon of animal life.     

High frequency of multiple paternity in the largest rookery of Mediterranean loggerhead sea turtles

Mating systems are a central component in the evolution of animal life histories and in conservation genetics. The patterns of male reproductive skew and of paternal shares in batches of offspring, for example, affect genetic effective population size. A prominent characteristic of mating systems of sea turtles seem to be a considerable intra- and interspecific variability in the degree of polyandry. Because of the difficulty of observing the mating behaviour of sea turtles directly in the open sea, genetic paternity analysis is particularly useful for gaining insights into this aspect of their reproductive behaviour. We investigated patterns of multiple paternity in clutches of loggerhead sea turtles in the largest Mediterranean rookery using four highly variable microsatellite loci. Furthermore, we tested for a relationship between the number of fathers detected in clutches and body size of females. More than one father was detected in the clutches of 14 out of 15 females, with two clutches revealing the contribution of at least five males. In more than half the cases, the contributions of different fathers to a clutch did not depart from equality. The number of detected fathers significantly increased with increasing female body size. This relationship indicates that males may prefer to mate with large, and therefore productive, females. Our results suggest that polyandry is likely to increase effective population size compared to a population in which females would mate with only one male; male reproductive contributions being equal.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5 end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.     

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.     

Growth hormone releasing effect of hpGRF-40 in rats at different time intervals following ablation of the mediobasal hypothalamus

We have investigated the effect of hypothalamo-pituitary disconnection in the rat on the growth hormone (GH) responsiveness to human pancreatic GH-releasing factor (hpGRF). Adult female rats, sham-operated (sham-op) or bearing a complete mechanical ablation of the mediobasal hypothalamus (MBH-A) were challenged, while under urethane anesthesia, with hpGRF-40 (20,100,500 ng/rat i.v.) at different time intervals after surgery. In sham-op rats only 500 ng/rat of hpGRF-40 stimulated GH release, while in 1-and 7-day MBH-A rats the stimulation also occurred with the lower hpGRF doses and the rise in plasma GH was greater than in sham-op controls. Twenty-one and 42 days after the placing of the lesions the GH response to hpGRF-40 was still present at the 500 ng/rat dose, though it was smaller than in sham-op controls. Evaluation of pituitary GH content demonstrated a progressive and rapid decline starting the first day after the placing of the lesions. These data indicate that GH responsiveness to hpGRF is: 1) enhanced in the anterior pituitary shortly after hypothalamo-pituitary disconnection and, 2) despite a striking reduction of the pituitary GH stores, it is maintained after these lesions.The physiologic growth hormone (GH) releaser in the rat is GH-releasing factor and, recently, a group of peptides has been characterized from human pancreatic tumors (hpGRFs) (1,2) which are potent and specific GH-releasers in both animals (3) and man (4). The availability of these peptides, which show a high degree of homology with the physiologic rat hypothalamic GRF (5), offers the unique opportunity to assess somatotrope responsiveness to GRF molecules in rats with hypothalamo-pituitary disconnection.In this study we have first evaluated the GH pituitary responsiveness to increasing doses of hpGRF-40 in rats following mechanical ablation of the mediobasal hypothalamus (6). These rats, by definition, lack the effect of both central nervous system (CNS) inhibitory (e.g. somatostatin) and stimulatory (e.g. GRF) influences to GH release. With the aim to ascertain how the lack of these two opposing inputs reflects on the secretory capacity of the somatotropes, we also investigated the GH response to hpGRF-40 at different time intervals after the lesioning. In a study in rats with electrolytic lesions of the ventromedial-arcuate region of the hypothalamus Tannenbaum et al (7) had shown persistence of the GH response to huge doses of a hpGRF analog.     

Tracking reveals behavioural coordination driven by environmental constraints in the Black-vented Shearwater Puffinus opisthomelas

During the breeding season, seabird foraging trips are constrained by nest attendance schedule and are necessarily colony centred. Oceanographic cues play a major role in the choice of foraging areas to minimize the time spent away from the nest. Here, we analysed the foraging tracks of Black-vented Shearwaters Puffinus opisthomelas during the incubation and chick-rearing periods of 2016 and 2017 at Isla Natividad (Mexico). We applied expectation-maximization binary clustering to track data to clusterize different behaviour patterns during foraging flights. We then applied binary generalized linear mixed models to characterize of foraging areas based on of environmental variables. We finally used kernel estimation techniques to describe main foraging areas. In 2016, breeding shearwaters used two core areas for foraging and resting on the water; the core area delineated by males was located northward from the colony in the Vizcaino Bay and the core area for females was located southward from the colony at the entrance of San Ignacio Lagoon. In 2017, males and females used the same areas with no evident segregation. Our study provided the first information on Black-vented Shearwater foraging areas during the breeding season and indicated that sexual segregation within coastal waters off the central Baja California Peninsula might be a foraging strategy during years of warmer ocean, likely less productive regimes. Factors including ocean-climate-mediated sexual segregation at sea, leading to interannual variation in foraging areas, should be considered when evaluating management actions intended to protect critical foraging habitats for Black-vented Shearwaters.