Osteopontin overexpression inhibits in vitro re-endothelialization via integrin engagement

The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via alpha(v) integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence (DeltaRGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and DeltaRGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via alpha(v) integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.     

Glutathione is recruited into the nucleus in early phases of cell proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.     

The expression of TRH, its receptors and degrading enzyme is differentially modulated in the rat limbic system during training in the Morris water maze

TRH administration induces arousal, improves cognition, and modulates glutamatergic and cholinergic transmission in hippocampal neurons. To study the possible involvement of TRH neurons in learning and memory processes, gene expression of TRH, its receptors, and pyroglutamyl peptidase (PPII), were measured in limbic regions of water-maze trained rats. Hypothalamus and amygdala showed changes related to the task but not specific to spatial learning while in hippocampus, pro-TRH and TRH-R1 mRNA levels were specifically increased in those animals trained to find a hidden platform. Variation of TRH content and mRNA levels of pro-TRH, TRH-R1, TRH-R2 and PPII are observed in conditions known to activate TRH hypophysiotropic neurons. Changes in some of these parameters could indicate the activation of TRHergic neurons and their possible involvement in some memory related process. Male Wistar rats were immersed (10 times) for 1, 3 or 5 days in a Morris water-maze containing, or not (yoked control) a platform and sacrificed 5, 30 and 60 min after last trial. TRH content and TSH serum levels were determined by radioimmunoassay; mRNA levels of pro-TRH, TRH-R1, TRH-R2, and PPII, by RT-PCR. Exclusive changes due to spatial training were observed in posterior hippocampus of rats trained for 5 days sacrificed after 60min: decreased TRH content and increased mRNA levels of pro-TRH and TRH-R1, particularly in CA3 region (measured by in situ hybridization). The hypothalamus-pituitary axis responded in both yoked and trained animals (increasing serum TSH levels and pro-TRH expression, due to swim-stress); in the amygdala of both groups, pro-TRH expression increased while diminished that of both receptors and PPII. Differential expression of these parameters suggests involvement of TRH hippocampal neurons in memory formation processes while changes in amygdala could relate to TRH anxiolytic role. The differential modulation in anterior and posterior portions of the hippocampus is discussed.     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.     

Social compatibility in a newly formed all‐male group of white crowned mangabeys (Cercocebus atys lunulatus)

Surplus males in primate captive populations are a common problem for zoos. Some captive breeding programs promote all‐male groups as an adequate option to house surplus males, but there have been few attempts to assess the feasibility of this management technique across primate species. The present study provides preliminary data regarding social compatibility within a newly formed all‐male group of four white crowned mangabeys (Cercocebus atys lunulatus). The study was conducted at the Valencia Zoo (Spain), where data on social behavior were collected over 6 months using continuous focal animal sampling for a total of 87 hr of observation. Results show that low intensity aggressive behaviors (facial threats) were expressed at high rates, whereas physical aggression (fights) rarely occurred. Aggression was more frequent among individuals belonging to the same age–gender class. Regarding affiliative behaviors, every individual actively sought proximity to all other group members through positive approaches, and although not all males carried out social grooming, every male was groomed by at least one group member. Our results suggest that the group was compatible socially because social relationships among the individuals were not neutral, and physical aggression occurred at low rates. The present study provides preliminary data supporting the feasibility of all‐male groups as a management option for surplus males in captive populations of white crowned mangabeys. Nevertheless, further studies are needed to be able to generalize both within and across species. Zoo Biol 0:1–7, 2007. © 2006 Wiley‐Liss, Inc.     

The alphaD-globin gene originated via duplication of an embryonic alpha-like globin gene in the ancestor of tetrapod vertebrates

Gene duplication is thought to play an important role in the co-option of existing protein functions to new physiological pathways. The globin superfamily of genes provides an excellent example of the kind of physiological versatility that can be attained through the functional and regulatory divergence of duplicated genes that encode different subunit polypeptides of the tetrameric hemoglobin protein. In contrast to prevailing views about the evolutionary history of the alpha-globin gene family, here we present phylogenetic evidence that the alpha(A)- and alpha(D)-globin genes are not the product of a single, tandem duplication of an ancestral globin gene with adult function in the common ancestor of extant birds, reptiles, and mammals. Instead, our analysis reveals that the alpha(D)-globin gene of amniote vertebrates arose via duplication of an embryonic alpha-like globin gene that predated the radiation of tetrapods. The important evolutionary implication is that the distinct biochemical properties of alpha(D)-hemoglobin (HbD) are not exclusively derived characters that can be attributed to a post-duplication process of neofunctionalization. Rather, many of the distinct biochemical properties of HbD are retained ancestral characters that reflect the fact that the alpha(D)-globin gene arose via duplication of a gene that had a larval/embryonic function. These insights into the evolutionary origin of HbD illustrate how adaptive modifications of physiological pathways may result from the retention and opportunistic co-option of ancestral protein functions.     

From forest to pasture: an evaluation of the influence of environment and biogeography on the structure of beetle (Scarabaeinae) assemblages along three altitudinal gradients in the Neotropical region

The objective of this study is to evaluate the effect of environmental (associated with the expansion of cattle ranching) and biogeographical factors on the diversity of dung beetle (Scarabaeinae) assemblages along three altitudinal gradients in the Neotropical region. One gradient is located in the Mexican Transition Zone, on the Cofre de Perote mountain, the other two are in the northern Andes (the Chiles Volcano and the Río Cusiana Basin). For the three gradients, the number of species and of individuals was similar in both forest and pasture, while species composition was different between habitats. On this mountain, species turnover in pastures was characterized by the addition of new species as altitude increased. In the northern Andes, species diversity was always greater in the forest than in the pasture, and species turnover between habitats was notably influenced by species loss with increasing altitude. As such the pasture fauna of the northern Andes was an impoverished derivative of the fauna present in the forests at the same altitude characterized by species of Neotropical affinity with a limited capacity for colonizing open, sunnier habitats. The opposite occurs in the areas used by cattle on the Cofre de Perote. This habitat has its own fauna, which is mainly comprised of Holarctic and Afrotropical species adapted to the prevailing environmental conditions of areas lacking arboreal vegetation. These results suggest that the impact on beetle communities caused by human activities can differ depending on the geographic position of each mountain and, particularly, the biogeographical history of the species assemblage that lives there.     

Diversity,geographic variation and conservationof the serpentine flora of Tuscany (Italy)

The specialised flora of serpentine outcrops in Tuscany (Italy) is analysed in terms of species richness and geographic variation, in order to identify main centres of diversity and provide a basis for conservation programmes. Five edaphic groups are distinguished, among which obligate endemics, serpentine-preferential taxa, facultative basiphilous and facultative calcifuge serpentinophytes. Relatively low diversity (87 taxa) and high taxonomic distinctiveness (28.7% of endemics plus preferentials) underscore the insular condition of the ophiolitic outcrops. Hemicryptophytes and chamaephytes are the dominat life-forms, in line with the mainly continental character revealed by the phytogeographical analysis. Presence/absence of the taxa in 10 serpentine island systems was assessed using literature and original field data. Number of species and surface of the areas are significantly correlated. Cluster analysis identifies five groups of areas, while ordination indicates the species which are more effective in determining the floristic differences among the areas. Cecina valley, Monte Ferrato, Murlo hills and upper Tiber valley are the main centres of endemism and taxonomic diversity. However, the positive relationship between floristic and geographic distances and the remarkable proportion of species with frequency < 50% highlights a considerable among-area variation. To ‘catch’ such variation, a network of distant protected sites appears more effective than to search for single areas with high diversity. At least one site in each of the five clusters should be included in a ‘Important Plant Area’ network which would ensure the conservation of such a peculiar component of the Italian vascular flora.     

Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis

The floral transition marks the switch from vegetative to reproductive growth, and is controlled by different pathways responsive to endogenous and exogenous cues. The developmental switch is accompanied by local changes in chromatin such as histone modifications. In this study we demonstrate large-scale reorganization of chromatin in rosette leaves during the floral transition. An extensive reduction in chromocenters prior to bolting is followed by a recovery of the heterochromatin domains after elongation of the floral stem. The transient reduction in chromocenters is a result of relocation away from chromocenters of methylated DNA sequences, 5S rDNA and interspersed pericentromeric repeats, but not of 45S rDNA or the 180-bp centromere tandem repeats. Moreover, fluorescence in situ hybridization analysis revealed decondensation of chromatin in gene-rich regions. A mutant analysis indicated that the blue-light photoreceptor CRYPTOCHROME 2 is involved in triggering chromatin decondensation, suggesting a light-signaling pathway towards large-scale chromatin modulation.