Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.     

The Influence of Chemical Stimuli from Predators on Precopulatory Pairing by the Amphipod,Gammarus pseudolimnaeus

Many amphipod crustaceans exhibit precopulatory mate guarding. Field samples of the amphipod Gammarus pseudolimnaeus indicated that pairs were positively size assortative. Receptive individuals readily formed pairs in the laboratory and the latency to formation of precopulatory pairs was decreased under threat of predation. In addition, females and, under conditions of extreme danger, males that formed pairs were significantly smaller when under the threat of predation. Amphipods distinguished between chemical stimuli (aquarium water) from predatory and nonpredatory fishes and between chemical cues from fish predators (trout) that had recently eaten conspecific amphipods vs. those fed a control diet of pelleted commercial fish food. These data indicate that chemical stimuli associated with predators can influence reproductive behavior of amphipods. The results also suggest the hypotheses that: 1. search time may be costly in terms of probability of predation; and 2. small pairs may be safer from predation than larger pairs.     

Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L.

The function of aspartic proteinases (EC 3.4.23) present in flowers of Cynara species is still unknown. Cardosin A, as a highly abundant aspartic proteinase from Cynara cardunculus L., a relative of the artichoke, is synthesised as a zymogen and subsequently undergoes proteolytic processing, yielding the mature and active enzyme. Here we report the study of the expression and localization of cardosin A, as a first approach to address the question of its physiological relevance. A polyclonal antibody specific for cardosin A was raised against a synthetic peptide corresponding to an amino acid sequence of the enzyme. This antibody was used to study the organ-specific, tissue-specific and subcellular localization of cardosin A by immunoblotting, tissue printing and immunogold electron microscopy. The results showed that expression of cardosin A is highly restricted to the pistils, and that the enzyme accumulates mainly in protein storage vacuoles of the stigmatic papillae. Cardosin A is also present, although much less abundantly, in the vacuoles of the cells of the epidermis of the style. In view of these results, the possible physiological roles of cardosin A are discussed, namely an involvement in defense mechanisms or pollen-pistil interaction, as well as in flower senescence. Received: 10 December 1996 / Accepted: 14 March 1997     

Vertical and spatial distribution of the near-shore littoral meroplankton off the Bay of Blanes (NW Mediterranean Sea)

The total number of meroplankton larvae collected off the Bayof Blanes (NW Mediterranean) on 12 April 1995 ranged from 113to 203 ind. m^–3. Bivalve veligers were the most abundant,followed by polychaete larvae, among which those from the hesionids,spionids and nephtyds were dominant Larval abundances were higherin the deeper half of the water column and decreased just nearthe bottom, except in the shallowest confined site.     

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.     

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.     

beta-xylosidase activity and expression of a beta-xylosidase gene during strawberry fruit ripening.

Strawberry fruit shows a marked softening during ripening and the process is associated with an increment of pectin solubility and a reduction of the molecular mass of hemicelluloses. In this work, we report the activity of beta-xylosidase and the expression of a beta-xylosidase gene in strawberry fruit. We have cloned a cDNA fragment encoding a putative beta-xylosidase (FaXyl1) from a cDNA library obtained from ripe strawberry fruit. The analysis of the deduced amino acid sequence revealed that FaXyl1 is closely related to other beta-xylosidases from higher plants. The expression of FaXyl1 was strongly associated to the receptacle tissue although a low expression level was detected in achenes and ovaries. The accumulation of FaXyl1 mRNA is ripening-related, starting in white fruit, reaching the maximum at 25-50% red fruit and decreasing thereafter. The total beta-xylosidase enzyme activity was detected in all ripening stages with the maximum in 25-50% red fruit. The low activity level detected in immature stages, where no expression of FaXyl1 was found, suggests the presence of other beta-xylosidases-like genes. Both the expression of FcaXyl1 and the total beta-xylosidase activity were down regulated by auxins, as occurs for most of the ripening-related processes in strawberry fruit. A putative role of FaXyl1 and beta-xylosidase is discussed.     

The specificity of alcohol dehydrogenase with cis-retinoids. Activity with 11-cis-retinol and localization in retina.

Studies in knockout mice support the involvement of alcohol dehydrogenases ADH1 and ADH4 in retinoid metabolism, although kinetics with retinoids are not known for the mouse enzymes. Moreover, a role of alcohol dehydrogenase (ADH) in the eye retinoid interconversions cannot be ascertained due to the lack of information on the kinetics with 11-cis-retinoids. We report here the kinetics of human ADH1B1, ADH1B2, ADH4, and mouse ADH1 and ADH4 with all-trans-, 7-cis-, 9-cis-, 11-cis- and 13-cis-isomers of retinol and retinal. These retinoids are substrates for all enzymes tested, except the 13-cis isomers which are not used by ADH1. In general, human and mouse ADH4 exhibit similar activity, higher than that of ADH1, while mouse ADH1 is more efficient than the homologous human enzymes. All tested ADHs use 11-cis-retinoids efficiently. ADH4 shows much higher k(cat)/K(m) values for 11-cis-retinol oxidation than for 11-cis-retinal reduction, a unique property among mammalian ADHs for any alcohol/aldehyde substrate pair. Docking simulations and the kinetic properties of the human ADH4 M141L mutant demonstrated that residue 141, in the middle region of the active site, is essential for such ADH4 specificity. The distinct kinetics of ADH4 with 11-cis-retinol, its wide specificity with retinol isomers and its immunolocalization in several retinal cell layers, including pigment epithelium, support a role of this enzyme in the various retinol oxidations that occur in the retina. Cytosolic ADH4 activity may complement the isomer-specific microsomal enzymes involved in photopigment regeneration and retinoic acid synthesis.     

Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates.

The role of the negative charge of the E139 side-chain of Anabaena Ferredoxin-NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side-chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge-reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side-chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild-type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.     

Radiation effects on oxidative metabolism in young and aged rat alveolar macrophages.

The effect of ionizing radiation on metabolic functions of alveolar macrophages (AM) have been well studied. However, variations associated to age have not been established yet. The aim of this work was to perform a comparative study on irradiated alveolar macrophages from young and aged rat lungs. Cell viability and occurrence of apoptosis as well as production of nitric oxide (NO), generation of superoxide anion (O2*-) and total antioxidant capacity were analyzed in vitro after exposure to gamma-irradiation with 10, 25, 50 and 75 Gy. Cell viability decreased only in the aged population at the higher doses. Morphological features of apoptosis were clearly evidenced in irradiated alveolar macrophages from aged animals although the DNA fragmentation assay for apoptosis showed no differences for either of the populations studied. NO production and total reactive antioxidant potential (TRAP) levels showed a dose-dependent modulation. Low radiation doses inhibited the production of NO and decreased TRAP levels whereas higher doses enhanced the NO production and increased the TRAP levels in both macrophage populations. Generation of O2*- was always higher in the aged population for all the doses assayed. We conclude that in vitro young alveolar macrophages exhibited higher radioresistance over the whole range of doses as compared to the aged macrophage population. Our results show that the aging process markedly affects the radioresistance of phagocytic cells. Therefore, immune defense and inflammatory response of lungs from aged patients should be considered when planning radiotherapy protocols.