The Generation of Active Oxygen Species Differs in Tobacco and Grapevine Mesophyll Protoplasts

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.     

Ammonium-induced increase in NADH-glutamate dehydrogenase activity is caused by de-novo synthesis of the α-subunit

When callus derived from shoot segments of Vitis vinifera L. was transferred to ammonium-containing medium the aminating activity of NAD(H)-glutamate dehydrogenase (GDH, EC 1.4.1.2) increased significantly. This increase in enzyme activity closely paralleled an increase in the protein of the GDH -subunit (43.0 kDa), as detected by sodium dodecyl sulfate (SDS) gel electrophoresis and Western-blotting. A similar correlation was observed between the deaminating activity and the -subunit (42.5 kDa) which both decreased during this treatment. Using [³⁵S]methionine and immunochemical detection it was shown that the rate of synthesis of the -subunit increased considerably in the ammonium-containing medium while there was no detectable synthesis of the -subunit. At the isoenzyme level, ammonium caused an increase in the de-novo synthesis and hence the activity staining of the more anodic isoenzymes, which are hexameric and consist mainly of -subunits. The results indicate that the increase in NADH-GDH specific activity was due to de-novo synthesis of the -subunit of GDH and the assembly of only the more anodic isoenzymes.Abbreviations GDH glutamate dehydrogenase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Physiological responses of beech and sessile oak in a natural mixed stand during a dry summer

Responses of CO2 assimilation and stomatal conductance to decreasing leaf water potential, and to environmental factors, were analysed in a mixed natural stand of sessile oak (Quercus petraea ssp. medwediewii) and beech (Fagus svlvatica L.) in Greece during the exceptionally dry summer of 1998. Seasonal courses of leaf water potential were similar for both species, whereas mean net photosynthesis and stomatal conductance were always higher in sessile oak than in beech. The relationship between net photosynthesis and stomatal conductance was strong for both species. Sessile oak had high rates of photosynthesis even under very low leaf water potentials and high air temperatures, whereas the photosynthetic rate of beech decreased at low water potentials. Diurnal patterns were similar in both species but sessile oak had higher rates of CO2 assimilation than beech. Our results indicate that sessile oak is more tolerant of drought than beech, due, in part, to its maintenance of photosynthesis at low water potential.     

Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation

BackgroundLeishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action.Methodology/Principal findingsWe tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes.Conclusions/SignificanceTPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF’s dual mode of action that encourages further studies.     

Polyamine anabolic/catabolic regulation along the woody grapevine plant axis

The distribution of the endogenous PA fractions throughout the entire perennial woody grapevine (Vitis vinifera L.) plant was studied, along with the expression profiles of the PA anabolic and catabolic genes and their substrates and secondary metabolites. Putrescine fractions increased with increasing leaf age, although the expression of its biosynthetic enzymes Arg and Orn decarboxylases decreased. Orn transport from young organs dramatically enhanced putrescine biosynthesis in older tissues, via the Orn decarboxylase pathway. S-adenosylmethionine decarboxylase and spermidine synthase genes were down-regulated during development in a tissue/organ-specific manner, as were spermidine and spermine levels. In contrast, amine oxidases, peroxidases and phenolics increased from the youngest to the fully developed vascular tissues; they also increased from the peripheral regions of leaves to the petioles. Hydrogen peroxide generated by amine oxidases accumulated for the covalent linkage of proteins via peroxidases during lignification. These results could be valuable for addressing further questions on the role of PAs in plant development.     

Nickel-quinolones interaction: Part 3 — Nickel(II) complexes of the antibacterial drug flumequine

Nickel(II) complexes with the first-generation quinolone antibacterial agent flumequine in the presence or absence of nitrogen donor heterocyclic ligands (4-benzylpyridine, pyridine, 2,2′-bipyridine or 1,10-phenanthroline) have been structurally characterized by physicochemical and spectroscopic techniques. The experimental data suggest that flumequine acts as deprotonated bidentate ligand coordinated to Ni(II) through the carboxylato and ketone oxygen atoms. The crystal structures of bis(4-benzylpyridine)bis(flumequinato)nickel(II) 2, (2,2′-bipyridine)bis(flumequinato)nickel(II) 4 and (1,10-phenanthroline)bis(flumequinato)nickel(II) 5 have been determined by X-ray crystallography and are the first crystal structures of flumequinato complexes reported. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes bind to CT DNA and bis(aqua)bis(flumequinato)nickel(II) exhibits the highest binding constant to CT DNA. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The cyclic voltammograms of the complexes recorded in DMSO solution and in 1/2 DMSO/buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that in the presence of CT DNA they bind to CT DNA by the intercalative binding mode. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values.     

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis

Hydrogen sulfide (H₂S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H₂S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H₂S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse^-/-) and wild-type mice with a physiological expression (Cse^+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse^+/+ donor; c) treatment with H₂S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H₂S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H₂S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H₂S higher and less than 5.3 μΜ (p = 7 x 10⁻⁶). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse^+/+ mice, whereas BMT increased the survival of Cse^-/- mice. Cse^-/- mice had increased pathogen loads compared to Cse^+/+ mice. Phagocytic activity of leukocytes from Cse^-/- mice was reduced but was restored after H₂S supplementation. An H₂S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H₂S is a potential independent parameter correlating with the outcome of P. aeruginosa. H₂S provides resistance to infection by MDR bacterial pathogens.     

Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB.

KorA and KorB proteins of IncP1 plasmid RK2 are encoded in the central control region (ccr) of the plasmid and act as global regulators of plasmid genes for replication, transfer and stable inheritance. KorA represses seven promoters on RK2, by binding to a defined operator site, OA, which always occurs in promoter regions. KorB recognises another operator, OB, which is found 12 times on the RK2 genome, but not always in promoter regions. At five of the KorA-regulated promoters, an OBsequence is also present. The presence of both KorA and KorB leads to severely decreased promoter activity. By measuring repression at different levels of KorA and KorB alone and in combination, we showed that there is at least 3. 4-fold co-operativity between them at korApin vivo. Testing the ability of previously isolated KorA mutants to act in a co-operative way in the presence of KorB in vivo or in vitro showed that the C-terminal part of KorA between amino acid positions 68 and 83 is required for this co-operativity. This region is part of a segment that is highly conserved between KorA and two other RK2 proteins, TrbA and KlcB. We propose that this conserved region may provide the basis for co-operativity with KorB either indirectly, by modulating DNA structure near the KorB binding site, or directly by serving as the "recognition" patch of each protein by KorB. It may thus serve as a key domain in allowing a sensitive response of the global circuits to changes in repressor concentration and thus modulation of replication, transfer and maintenance.