ND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed.     

Anti-coagulant rodenticide binding properties of human serum albumin: a biochromatographic approach

In this paper, the anti-coagulant rodenticide-human serum albumin (HSA) binding was investigated using a perturbation method to calculate the solute distribution isotherms. It was shown that rodenticide can bound either on the benzodiazepine HSA site with low affinity (site I) or on the warfarin HSA site with high affinity (site II). The thermodynamic parameters of this association were calculated for the two HSA binding sites. For the site II, the rodenticide-HSA association was governed enthalpically whereas for the site I, this one was driven entropically. Moreover, the role of the magnesium (Mg(2+)) and calcium (Ca(2+)) on this association was carried out. It was clearly demonstrated that the rodenticide affinity for the site I was not affected by modifying the bulk solvent surface tension whereas for the site II the association constant increased strongly with the Mg(2+) or the Ca(2+) concentration in the bulk solvent. These results showed that the rodenticide-HSA affinity and thus the rodenticide toxicological effect depends on the Mg(2+) or Ca(2+) concentration.     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Dynamics of RecA filaments on single-stranded DNA

RecA, the key protein in homologous recombination, performs its actions as a helical filament on single-stranded DNA (ssDNA). ATP hydrolysis makes the RecA–ssDNA filament dynamic and is essential for successful recombination. RecA has been studied extensively by single-molecule techniques on double-stranded DNA (dsDNA). Here we directly probe the structure and kinetics of RecA interaction with its biologically most relevant substrate, long ssDNA molecules. We find that RecA ATPase activity is required for the formation of long continuous filaments on ssDNA. These filaments both nucleate and extend with a multimeric unit as indicated by the Hill coefficient of 5.4 for filament nucleation. Disassembly rates of RecA from ssDNA decrease with applied stretching force, corresponding to a mechanism where protein-induced stretching of the ssDNA aids in the disassembly. Finally, we show that RecA–ssDNA filaments can reversibly interconvert between an extended, ATP-bound, and a compressed, ADP-bound state. Taken together, our results demonstrate that ATP hydrolysis has a major influence on the structure and state of RecA filaments on ssDNA.     

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

Fibrillation of transferrin

BackgroundThe nature of fibrillar deposits from aqueous solutions of human serum and recombinant human transferrin on mica and carbon-coated formvar surfaces has been investigated.Methods and ResultsAtomic force microscopy showed that the deposition of recombinant transferrin onto the hydrophilic surface of mica resulted in the formation of a monolayer-thick film composed of conformationally-strained flattened protein molecules. Elongated fibres developed on top of this layer and appeared to be composed of single proteins or small clusters thereof. Monomeric and dimeric transferrins were separated by gel permeation chromatography and their states of aggregation confirmed by mass spectrometry and dynamic light scattering. Transmission electron-microscopy showed that dimeric transferrin, but not monomeric transferrin, deposited on carbon-coated formvar grids forms rounded (circular) structures ca. 250 nm in diameter. Small transferrin fibrils ca. 250 nm long appeared to be composed of smaller rounded sub-units. Synchrotron radiation-circular dichroism and, Congo red and thioflavin-T dye-binding experiments suggested that transferrin aggregation in solution does not involve major structural changes to the protein or formation of classical β-sheet amyloid structures. Collisional cross sections determined via ion mobility–mass spectrometry showed little difference between the overall protein shapes of apo- and holo-transferrin in the gas phase.General significanceThe possibility that transferrin deformation and aggregation are involved in neurological disorders such as Parkinson's and Alzheimer's disease is discussed. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.     

Can eggs in a cavity be a female secondary sexual signal? Male nest visits and modelling of egg visual discrimination in blue tits

Eggshell colouration is thought to function as a female-specific secondary sexual trait. While tests of this idea are rapidly accumulating in cavity-nesting birds, some fundamental underlying assumptions remain rarely investigated: namely, can males see eggshell coloration and perceive colour differences between the eggs of different females? We tested these two key assumptions in a natural population of blue tits (Cyanistes caeruleus). Using transponders, we tracked male nest visits and found that all males visited their nest-boxes while eggs were present and often visually accessible. Interestingly, some males also visited neighbouring nests. We then tested whether birds could detect eggshell coloration using models of avian colour vision; models were performed with and without limitations on visual performance owing to dim light. Both models found that differences in eggshell brightness were often easier to discriminate than differences in colour; there was more contrast in white eggshell background between clutches than within and its contrast against nest background was repeatable within clutches, suggesting these features could act as signals. Yet, the detectability of these contrasts depended entirely on model assumptions of visual limitations. Consequently, we need a better understanding of underlying visual mechanisms in dim-light environments and behavioural discrimination experiments before confirming the signalling potential of eggshell coloration.     

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.