Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.     

A high dose of long term treatment with deprenyl loses its effect on antioxidant enzyme activities as well as on survivals of Fischer-344 rats

The survival rate of male Fischer-344/Du rats treated chronically with high doses of deprenyl was investigated. Eighteen month old rats were treated with 1 mg/kg s.c. deprenyl 3 times per week for 13 months. At the age of 31 months, treated rats showed a greater mortality rate with three of 12 rats surviving, while in saline-treated control animals seven of 12 animals survived. No significant differences in superoxide dismutase (SOD) or catalase (CAT) activities in brain regions of control and treated animals were seen at 31 months of age. In contrast, when 27 month old rats were treated in the same manner for one month, significant increases in SOD (both Cu,Zn- and Mn-) and CAT activities were found in substantia nigra, striatum and cerebral cortex, but not in hippocampus. This effect was produced with a wide range of deprenyl doses (0.25-2 mg/kg, but not 4 mg/kg). Although a causal relationship between the two different effects of the drug, i.e. 1) increases in antioxidant enzyme activities and 2) the prolongation of survival of animals, has not been directly demonstrated, the loss of both effects with the high dose of the drug in the present experiment may be taken as circumstantial evidence for their causal relationship.     

Inactivation of Mitochondrial Permeability Transition Pore by Octylguanidine and Octylamine

Mitochondrial permeability transition occurs through a Ca²⁺-dependent opening of atransmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase(ANT). In this work, we induced permeability transition by adding 0.5 M carboxyatractyloside.The process was evaluated analyzing Ca²⁺ efflux, a drop in transmembrane electric gradient,and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at theconcentration of 100 M, inhibited, almost completely, nonspecific membrane permeability.Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to doso. The inhibition was reversed after the addition of 40 mM Li⁺, Na⁺ K⁺,Rb⁺, or Cs⁺; K⁺ wasthe most effective. We propose that the positive charge of the amines interact with negativecharges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetratesinto the hydrophobic milieu of the inner membrane, fixing the reagent.     

Quantification of the phosphorus released by zooplankton in an oligotrophic lake (La Caldera, Spain): regulating factors and adjustment to theoretical models

Using an in situ approach, we have evaluated the phosphorusinputs from zooplankton in a high-mountain oligotrophic lake.Values of the specific gross release rate (SGRR) fluctuatedbetween 0.2 and 2.9 µg P mg^–1 dry weight h^–1,and were higher when the nauplii of Mixodiaptomus laciniatusdominated the zooplankton community. The rate of P recyclingby the zooplankton was high, reaching 1.6 µg P l^–1day^–1, and showed a highly consistent seasonal patternfrom one year to the next, with maxima in midsummer. Zooplanktonsize accounted for as much as 85% of the variance obtained inthe measurements of the specific rate of P release, while otherfactors, such as the quality or quantity of food, did not significantlyinfluence the SGRR changes. Among the models tested, only theone proposed by Peters (Int, Ver. Theor. Angew. Limnol. Verh.,19, 273–279, 1975) was useful for predictions in thissystem. The stoichiometric model of Hessen and Andersen (Arch.Hydrobiol., 35, 111–120, 1992), applied in this oligotrophicsystem, adequately predicted the phyto- and zooplankton dynamics,whereas the values of P release estimated using this model werefar higher than the excretion rates obtained experimentally.These differences were related to the type of egestion (formationof faecal pellets) of metazooplankton and to the relative importanceof the food resistance to digestion. We believe that in communitieswhere copepods constitute a substantial percentage of the zooplankton,an evaluation of the P release which is readily available [solublereactive phosphate (SRP), total dissolved phosphate (TDP)] toalgae and bacteria would not fit the predictions of generalmodels of mass balance; under these circumstances, assimilationefficiency proves to be the key parameter for predicting thereadily available P (excreted P).     

Biophysical Studies on BEX3, the p75NTR-Associated Cell Death Executor,Reveal a High-Order Oligomer with Partially Folded Regions

BEX3 (Brain Expressed X–linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55–120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data.     

Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi

Pyridoxinamine 5''-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5''-phosphate or pyridoxamine 5''-phosphate to form pyridoxal 5''-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.     

Policy Strategies to Promote Eco‐Innovation

This article elaborates an integrated policy framework, including policy features and specific measures that can be implemented to mitigate the barriers to eco‐innovations. We claim that factors inhibiting the development and uptake of eco‐innovations are multifaceted and stem from different sources. This systems view, which highlights the multilayered conception of the obstacles to eco‐innovation, calls for a combination of environmental and technology policies adapted to the different barriers and characteristics of the technologies. Drawing on evolutionary economics, the article sheds light on the appropriate combinations of framework conditions and instruments that are most effective to promote eco‐innovation, considering different barriers and eco‐innovation types (process/product, mature/immature, and radical/incremental).