Virtual plants: modelling as a tool for the genomics of tolerance to water deficit

Modelling can simulate the responses of virtual plants carrying diverse combinations of alleles under different scenarios of abiotic stress. The main difficulty is mathematically expressing the genetic variability of responses to environmental conditions. Modelling via gene regulatory networks is not feasible for such complex systems, but plants can be modelled using response curves to environmental conditions that are 'meta mechanisms' at plant level. Each genotype is represented by a set of response parameters that are valid under a wide range of conditions. Transgenesis of one function experimentally affected one response parameter only. Transgenic plants or plants carrying any combination of quantitative trait loci might therefore be simulated and tested under different climatic scenarios, before genetic manipulations are performed.     

Dimethylsulfoxide oxidizes glutathione in vitro and in human erythrocytes: kinetic analysis by 1H NMR

The interaction of dimethylsulfoxide (Me2SO) with glutathione was investigated under non-equilibrium conditions in solution using 1H NMR and in intact erythrocytes using 1H spin-echo NMR. In solution the reaction was observed to follow second-order kinetics (Rate = k1[glutathione][Me2SO]) at 300 K pH 7.4, k(sol) = 4.7 x 10(-5)mol(-1)L(1)s(-1). In intact erythrocytes the rate constant for the cellular environment, k(cell), was found to be slightly larger at 8.1 x 10(-5)mol(-1)L(1)s(-1). Furthermore, the reaction of Me2SO with erythrocyte glutathione showed a biphasic dependence on the Me2SO concentration, with little oxidation of glutathione occurring until the Me2SO concentration exceeded 0.5 molL(-1). The results suggest that at lower concentrations, Me2SO can be effectively removed, most probably by reaction with glutathione, which is regenerated by glutathione reductase, although preferential reaction with other cellular components (e.g., membrane or cellular thiols) cannot be ruled out. Thus the concentrations of Me2SO that are commonly used in cryopreservation of mammalian cells ( approximately 1.4 molL(-1)) can cause oxidation of intracellular glutathione.     

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.     

NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.     

Suppression of CD4+ T lymphocyte effector functions by CD4+CD25+ cells in vivo

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-gamma production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-gamma as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.     

Atg1 allows second-signaled autophagic cell death in Dictyostelium

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.     

No evidence for an association between the -871 T/C promoter polymorphism in the B-cell-activating factor gene and primary Sjögren's syndrome

Polyclonal B cell activation might be related to pathogenic over-expression of B-cell-activating factor (BAFF) in primary Sjögren's syndrome (pSS) and other autoimmune diseases. We therefore investigated whether BAFF over-expression in pSS could be a primary, genetically determined event that leads to the disease. The complete BAFF gene was sequenced in Caucasian pSS patients and control individuals. The only single nucleotide polymorphism frequently observed, namely -871 T/C in the promoter region, was then genotyped in 162 French patients with pSS and 90 French control individuals. No significant differences in allele (T allele frequency: 49.7% in patients with pSS versus 50% in controls; P = 0.94) and genotype frequencies of BAFF polymorphism were detected between pSS patients and control individuals. BAFF gene polymorphism was not associated with a specific pattern of antibody secretion either. T allele carriers had significantly increased BAFF protein serum levels (mean values of 8.6 and 5.7 ng/ml in patients with TT and TC genotypes, respectively, versus 3.3 ng/ml in patients with CC genotype; P = 0.01), although no correlation was observed between BAFF polymorphism and mRNA level. In conclusion, BAFF gene polymorphism is neither involved in genetic predisposition to pSS nor associated with a specific pattern of antibody production.     

Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.