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1.  Mechanisms of disruption of meristematic competence by microgravity in Arabidopsis seedlings 
Plant Signaling & Behavior  2014;9:e28289.
Experiments performed in actively proliferating plant cells both in space and simulated microgravity have evidenced a common effect: cell proliferation appears enhanced whereas cell growth is depleted. Coordination of cell growth and proliferation, called meristematic competence, is a major feature of meristematic cells and its disruption may lead to important alterations in the developmental pattern of the plant. Auxin is known to be a mediator of the transduction of the gravitropic signal and a regulator of the rates of growth and proliferation in meristematic cells, as well as of their further differentiation. Therefore, gravity sensing, gravitropism, auxin levels, and meristematic competence are mutually interrelated. However, our experiments in simulated microgravity, using both mechanical and magnetic levitation technologies, have revealed that this interdependence is neither strict nor univocal and may include additional factors and mechanisms. Available data indicate that altered gravity may affect cell growth and proliferation by mechanisms alternative to the transduction of the gravitropic signal perceived by columella cells in the root tip. These mechanisms would include gravity sensing independent from statolith displacement and transduction mediators other than polar auxin transport.
PMCID: PMC4091523  PMID: 24614101
cell cycle; ribosome biogenesis; nucleolus; graviperception; simulated microgravity; Arabidopsis
2.  Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station 
PLoS ONE  2014;9(3):e91814.
The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.
PMCID: PMC3950288  PMID: 24618597
3.  Constitutive Phosphorylation of Interferon Receptor A-Associated Signaling Proteins in Systemic Lupus Erythematosus 
PLoS ONE  2012;7(7):e41414.
Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.
Methodology/Principal Findings
Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.
These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.
PMCID: PMC3408474  PMID: 22859983
4.  Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana 
PLoS Genetics  2010;6(11):e1001225.
In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.
Author Summary
Chromatin remodeling plays a central role in controlling gene expression in all eukaryotic organisms. Chromatin can be found in a repressive or transcriptionally inactive state (heterochromatin) or in a more permissive or transcriptionally active state (euchromatin). The building block of chromatin is the nucleosome, which consists of four histones, H2A, H2B, H3, and H4, surrounded by 147 base pairs of DNA. In addition, a linker histone H1 directs the path of DNA between adjacent nucleosomes to form the chromatin fiber. Chromatin compaction also depends on DNA methylation and on a number of histone modifications, including methylation and acetylation of histone tails. However, other non-histone proteins are required to direct chromatin structure and remodeling. Nucleolin is a major nucleolar protein involved not only in rRNA transcription and processing of 45S pre–rRNA transcribed by RNA Pol I, but also in the control of RNA pol II transcription in the nucleoplasm. Through genetic, molecular, and immunocytological approaches, we studied the role of this protein in vivo in controlling rRNA chromatin structure and the expression of hundreds of clustered rRNA genes using the model plant Arabidopsis thaliana.
PMCID: PMC2991258  PMID: 21124873
5.  Microgravity environment uncouples cell growth and cell proliferation in root meristematic cells 
Plant Signaling & Behavior  2010;5(2):176-179.
Experiments performed in space have evidenced that, in root meristematic cells, the absence of gravity results in the uncoupling of cell growth and cell proliferation, two essential cellular functions that support plant growth and development, which are strictly coordinated under normal ground gravity conditions. In space, cell proliferation appears enhanced whereas cell growth is depleted. Since coordination of cell growth and proliferation is a major feature of meristematic cells, the observed uncoupling is a serious stress condition for these cells producing important alterations in the developmental pattern of the plant. Auxin plays a major role in these processes both by assuring the coupling of cell growth and proliferation under normal conditions and by exerting a decisive influence in the uncoupling under altered gravity conditions. Auxin is a mediator of the transduction of the gravitropic signal and its distribution in the root is altered subsequent to a change in the gravity conditions. This altered distribution may produce changes in the expression of specific growth coordinators leading to the alteration of cell cycle and protein synthesis. Therefore, available data indicate that the effects of altered gravity on cell growth and proliferation are the consequence of the transduction of the gravitropic signal perceived by columella cells, in the root tip.
PMCID: PMC2884128  PMID: 20173415
cell cycle; ribosome biogenesis; nucleolus; auxin efflux; graviperception; space flight; arabidopsis
6.  Relationship Between the Plasma Concentration of C-Reactive Protein and Severity of Peripheral Arterial Disease 
To determine whether the increase in plasma levels of C-Reactive Protein (CRP), a non-specific reactant in the acute-phase of systemic inflammation, is associated with clinical severity of peripheral arterial disease (PAD).
Methods and Results:
This is a cross-sectional study at a referral hospital center of institutional practice in Madrid, Spain. A stratified random sampling was done over a population of 3370 patients with symptomatic PAD from the outpatient vascular laboratory database in 2007 in the order of their clinical severity: the first group of patients with mild chronological clinical severity who did not require surgical revascularization, the second group consisted of patients with moderate clinical severity who had only undergone only one surgical revascularization procedure and the third group consisted of patients who were severely affected and had undergone two or more surgical revascularization procedures of the lower extremities in different areas or needed late re-interventions. The Neyman affixation was used to calculate the sample size with a fixed relative error of 0.1. A homogeneity analysis between groups and a unifactorial analysis of comparison of medians for CRP was done. The groups were homogeneous for age, smoking status, Arterial Hypertension HTA, diabetes mellitus, dyslipemia, homocysteinemia and specific markers of inflammation. In the unifactorial analysis of multiple comparisons of medians according to Scheffé, it was observed that the median values of CRP plasma levels were increased in association with higher clinical severity of PAD (3.81 mg/L [2.14–5.48] vs. 8.33 [4.38–9.19] vs. 12.83 [9.5–14.16]; p < 0.05) as a unique factor of tested ones.
Plasma levels of CRP are associated with not only the presence of atherosclerosis but also with its chronological clinical severity.
PMCID: PMC2872580  PMID: 20508761
C-reactive protein; peripheral arterial disease; inflammatory origen
7.  Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis 
Molecular Biology of the Cell  2007;18(2):369-379.
Nucleolin is one of the most abundant protein in the nucleolus and is a multifunctional protein involved in different steps of ribosome biogenesis. In contrast to animals and yeast, the genome of the model plant Arabidopsis thaliana encodes two nucleolin-like proteins, AtNUC-L1 and AtNUC-L2. However, only the AtNUC-L1 gene is ubiquitously expressed in normal growth conditions. Disruption of this AtNUC-L1 gene leads to severe plant growth and development defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer region decondensation, and affects the accumulation levels of pre-rRNA precursors. Remarkably, in Atnuc-L1 plants the AtNUC-L2 gene is activated, suggesting that AtNUC-L2 might rescue, at least partially, the loss of AtNUC-L1. This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.
PMCID: PMC1783796  PMID: 17108323

Results 1-7 (7)