Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

igments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 132 OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat (Triticum aestivum L. cv. Arina) plants. The segments were floated on H2O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m−2 s−1) in ambient air and in CO2-depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin-dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS-PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO2-depleted air. However, little effect of CO2 on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO2 was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO2. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO2-depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.

Protein Hydrolysis and Nitrogen Remobilisation in Plant Life and Senescence

In plant cells, as in all other cells, proteins are submitted to permanent turnover, and the intracellular content of a given protein depends on its rate of both synthesis and degradation. The life time of most proteins is shorter than that of the cell. Thus, in young leaves of Lemna minor, the average half-life of protein was estimated to be 7 days, and it was shorter under stress conditions (Davies 1982). Such observations mean that nitrogen and amino acid fluxes are both cylic and permanent. Although protein turnover may appear wasteful, in terms of energy, numerous studies have shown that proteolysis provides multiple functions in cell physiology, and is an essential regulatory mechanism of cell metabolism and development.

Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves

Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.

Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris (L.) leaves

Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713--1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.

Fractals for physicians

There is increasing interest in the study of fractals in medicine. In this review, we provide an overview of fractals, of techniques available to describe fractals in physiological data, and we propose some reasons why a physician might benefit from an understanding of fractals and fractal analysis, with an emphasis on paediatric respiratory medicine where possible. Among these reasons are the ubiquity of fractal organisation in nature and in the body, and how changes in this organisation over the lifespan provide insight into development and senescence. Fractal properties have also been shown to be altered in disease and even to predict the risk of worsening of disease. Finally, implications of a fractal organisation include robustness to errors during development, ability to adapt to surroundings, and the restoration of such organisation as targets for intervention and treatment.

Genome-wide association study of intracranial aneurysm identifies three new risk loci

Saccular intracranial aneurysms are balloon-like dilations of the intracranial arterial wall; their hemorrhage commonly results in severe neurologic impairment and death. We report a second genome-wide association study with discovery and replication cohorts from Europe and Japan comprising 5,891 cases and 14,181 controls with approximately 832,000 genotyped and imputed SNPs across discovery cohorts. We identified three new loci showing strong evidence for association with intracranial aneurysms in the combined dataset, including intervals near RBBP8 on 18q11.2 (odds ratio (OR) = 1.22, P = 1.1 x 10(-12)), STARD13-KL on 13q13.1 (OR = 1.20, P = 2.5 x 10(-9)) and a gene-rich region on 10q24.32 (OR = 1.29, P = 1.2 x 10(-9)). We also confirmed prior associations near SOX17 (8q11.23-q12.1; OR = 1.28, P = 1.3 x 10(-12)) and CDKN2A-CDKN2B (9p21.3; OR = 1.31, P = 1.5 x 10(-22)). It is noteworthy that several putative risk genes play a role in cell-cycle progression, potentially affecting the proliferation and senescence of progenitor-cell populations that are responsible for vascular formation and repair.

Disabling c-Myc in childhood medulloblastoma and atypical teratoid/rhabdoid tumor cells by the potent G-quadruplex interactive agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex-forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC(50), 0.25-0.39 micromol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc-dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.

Telomere length in human natural killer cell subsets

Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. In the elderly, the cytotoxic function of NK cells is often compromised. Telomeres progressively shorten with each cell division and with age in most somatic cells eventually leading to chromosomal instability and cellular senescence. We studied the telomere length in NK cell subsets isolated from peripheral blood using "flow FISH," a method in which the hybridization of telomere probe in cells of interest is measured relative to internal controls in the same tube. We found that the average telomere length in human NK cells decreased with age as was previously found for human T lymphocytes. Separation of adult NK cells based on CD56 and CD16 expression revealed that the telomere length was significantly shorter in CD56(dim)CD16(+) (mature) NK cells compared to CD56(bright)CD16(-) (immature) NK cells from the same donor. Furthermore, sorting of NK cells based on expression of activation markers, such as NKG2D and LFA-1, revealed that NK cells expressing these markers have significantly shorter telomeres. Telomere fluorescence was very heterogeneous in NK cells expressing CD94, killer inhibitory receptor (KIR), NKG2A, or CD161. Our observations indicate that telomeric DNA in NK cells is lost with cell division and with age similar to what has been observed for most other hematopoietic cells. Telomere attrition in NK cells is a plausible cause for diminished NK cell function in the elderly.

Methodical study of nitrous oxide eddy covariance measurements using quantum cascade laser spectrometery over a Swiss forest

Nitrous oxide fluxes were measured at the Lägeren CarboEurope IP flux site over the multi-species mixed forest dominated by European beech and Norway spruce. Measurements were carried out during a four-week period in October–November 2005 during leaf senescence. Fluxes were measured with a standard ultrasonic anemometer in combination with a quantum cascade laser absorption spectrometer that measured N2O, CO2, and H2O mixing ratios simultaneously at 5 Hz time resolution. To distinguish insignificant fluxes from significant ones it is proposed to use a new approach based on the significance of the correlation coefficient between vertical wind speed and mixing ratio fluctuations. This procedure eliminated roughly 56% of our half-hourly fluxes. Based on the remaining, quality checked N2O fluxes we quantified the mean efflux at 0.8±0.4 μmol m−2 h−1 (mean ± standard error). Most of the contribution to the N2O flux occurred during a 6.5-h period starting 4.5 h before each precipitation event. No relation with precipitation amount could be found. Visibility data representing fog density and duration at the site indicate that wetting of the canopy may have as strong an effect on N2O effluxes as does below-ground microbial activity. It is speculated that above-ground N2O production from the senescing leaves at high moisture (fog, drizzle, onset of precipitation event) may be responsible for part of the measured flux.

Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5

BACKGROUND: Oxidized low density lipoprotein (oxLDL) has been shown to induce apoptosis and senescence of endothelial progenitor cells (EPC). In the present study, we hypothesized that even sub-apoptotic concentrations of oxLDL impair the angiogenic potential of EPC and investigated if this effect is mediated by affecting adhesion and incorporation. METHODS: A co-culture system of human microvascular endothelial cells and EPC was used to study the effect of sub-apoptotic concentrations of native (nLDL) and oxLDL on cell-cell interaction. The expression and the functional role of angiogenic adhesion molecules and integrins was monitored by FACS and neutralizing assay, respectively. RESULTS: We observed an inhibition of tube formation and impairment of EPC integration into the vascular network of mature endothelial cells by oxLDL. In contrast, nLDL did not affect angiogenic properties of EPC. Incubation of EPC with sub-apoptotic oxLDL concentrations significantly decreased E-selectin and integrin alpha(v)beta(5) expression (37.6% positive events vs. 71.5% and 24.3% vs. 49.9% compared to control culture media without oxLDL). Interestingly, expression of alpha(v)beta(3), VE-cadherin and CD31 remained unchanged. Blocking of E-selectin and integrin alpha(v)beta(5) by neutralizing antibody effectively inhibited adhesion of EPC to differentiated endothelial cells (56.5% and 41.9% of control; p<0.001). CONCLUSION: In conclusion, oxidative alteration of LDL impairs angiogenic properties of EPC at sub-apoptotic levels by downregulation of E-selectin and integrin alpha(v)beta(5), both substantial mediators of EPC-endothelial cell interaction.

Selection and hydroponic growth of bread wheat cultivars for bioregenerative life support systems

As part of the ESA-funded MELiSSA program, the suitability, the growth and the development of four bread wheat cultivars were investigated in hydroponic culture with the aim to incorporate such a cultivation system in an Environmental Control and Life Support System (ECLSS). Wheat plants can fulfill three major functions in space: (a) fixation of CO2 and production of O2, (b) production of grains for human nutrition and (c) production of cleaned water after condensation of the water vapor released from the plants by transpiration. Four spring wheat cultivars (Aletsch, Fiorina, Greina and CH Rubli) were grown hydroponically and compared with respect to growth and grain maturation properties. The height of the plants, the culture duration from germination to harvest, the quantity of water used, the number of fertile and non-fertile tillers as well as the quantity and quality of the grains harvested were considered. Mature grains could be harvested after around 160 days depending on the varieties. It became evident that the nutrient supply is crucial in this context and strongly affects leaf senescence and grain maturation. After a first experiment, the culture conditions were improved for the second experiment (stepwise decrease of EC after flowering, pH adjusted twice a week, less plants per m2) leading to a more favorable harvest (higher grain yield and harvest index). Considerably less green tillers without mature grains were present at harvest time in experiment 2 than in experiment 1. The harvest index for dry matter (including roots) ranged from 0.13 to 0.35 in experiment 1 and from 0.23 to 0.41 in experiment 2 with modified culture conditions. The thousand-grain weight for the four varieties ranged from 30.4 to 36.7 g in experiment 1 and from 33.2 to 39.1 g in experiment 2, while market samples were in the range of 39.4–46.9 g. Calcium levels in grains of the hydroponically grown wheat were similar to those from field-grown wheat, while potassium, magnesium, phosphorus, iron, zinc, copper, manganese and nickel levels tended to be higher in the grains of experimental plants. It remains a challenge for future experiments to further adapt the nutrient supply in order to improve senescence of vegetative plant parts, harvest index and the composition of bread wheat grains.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

AIMS:Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS:Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION:Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.