The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Plant communities of the montane mesophilous grasslands (Polygono bistortae–Trisetion flavescentis alliance) in central Europe: Formalized classification and syntaxonomical revision

Abstract

The research conducted here presents a syntaxonomical revision of the montane mesophilous meadows of the Polygono bistortae-Trisetion flavescentis alliance in central Europe – the Slovak part of the western and eastern Carpathians. These typical semi-natural grasslands occur mainly as small islands over the calcareous bedrocks. Associations of this alliance have tight relationships to the Arrhenatherion, Mesobromion and Nardo-Agrostion alliances. Formal definitions based on combination of the species groups were used regarding the diversity and geographical range of the Polygono bistortae-Trisetion flavescentis alliance. Following the formal definitions from the eight associations reported previously for Slovakia, only four of them can be placed within the Polygono bistortae-Trisetion flavescentis: Campanulo glomeratae-Geranietum sylvatici, Geranio sylvatici-Trisetetum, Crepido mollis-Agrostietum capillaries and Geranio-Alchemilletum crinitae. A comparison of traditional and formalized classification, and the advantages/disadvantages of the formalized classification are discussed.     

Effects of soil water temperature on root hydraulic resistance of six species of Iberian pines

Abstract

The Iberian Peninsula hosts six native pine species, which are distributed according to an altitudinal gradient from coastal to mountain areas, close to 1000 m a.s.l. Root hydraulic responses are the key factors of spatial segregation of trees in response to environmental factors such as temperature and water availability, and they will be a determinant of future population and species spatial dynamics in a changing climate scenario. Root hydraulic responses to soil water temperatures ranging from 30°C to 0°C were compared for young plants of these six aforementioned species. Hydraulic resistance (Rh) increased for all species in response to temperature decrease. Mountain pines showed higher Rh values than coastal pines at all temperatures, and showed a more prompt and marked hydraulic response when temperatures dropped down. Data point out that mountain pines display a clear mechanism to avoid cold embolism and secondary water stress, while coastal species have a limited responsiveness to temperature changes due to scarce hydraulic regulation. These differences in hydraulic behaviour support the spatial segregation between mountain and coastal pines in the Iberian Peninsula, and will be one of the factors at the basis of the future shifts of species and populations that will be associated to climate change.     

Antimicrobial activity of various cationic molecules on foodborne pathogens

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.     

Antarctic scallop (Adamussium colbecki) annual growth rate at Terra Nova Bay

Adamussium colbecki is one of the most well-studied Antarctic molluscs. However, information on its growth rate is currently based on estimates from mark and recapture experiments and from growth-ring analyses. This paper provides the first estimates of the growth pattern of this scallop throughout a year, for individuals maintained under both natural field and laboratory conditions. Results show size-related differences in growth rate, both in the field and in aquaria, with scallops in Terra Nova Bay growing faster than those kept in aquaria. Growth performances were lower than those reported in the literature.     

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.     

Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance

The paucity of investigations on the presence of artificial radionuclides and their bioaccumulation in Antarctic fauna is due to the erroneous belief that this area is pristine. We report evidence that significant levels of the artificial radionuclides Sr-90, Cs-137, Am-241 and plutonium isotopes can be found in sponges, bivalves, krill and demersal fish fauna of Terra Nova Bay (Ross Sea), sometimes with a seasonal pattern. Increasing concentrations of Cs-137 were detected in the bivalve Adamussium colbecki (Antarctic scallop) during austral summer months, as a result of major trophic activity and changes in metabolic rates. Bioconcentration factors for artificial radionuclides in different Antarctic species are presented and discussed in relation to their different trophic strategies. Unexpectedly high radiocesium bioconcentration factors determined in bivalves suggested the particular role played by filter feeding in bioaccumulation, particularly in summer when radionuclide bioavailability is enhanced. The feeding preference of the trematomiid fish Trematomus bernacchii for the scallop A. colbecki is confirmed, not only by fish gut content analyses, but also through radiometric results. Transuranics bioaccumulation by sensitive species allowed some interesting comparisons on the different plutonium contamination of the southern hemisphere with respect to the northern one. Accepted: 25 April 2000