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The HIV reservoir is the main barrier to eradicating HIV infection, and resting memory CD4 T (Trm) cells are one of the most relevant cellular component harboring latent proviruses. This is the first study analyzing the transcriptional profile of Trm cells, in two well-characterized groups of HIV patients with distinct mechanisms of viral replication control (spontaneous versus treatment-induced). We use a systems biology approach to unravel subtle but important differences in the molecular mechanisms operating at the cellular level that could be associated with the host's ability to control virus replication and persistence. Despite the absence of significant differences in the transcriptome of Trm cells between Elite Controllers (ECs) and cART-treated (TX) patients at the single gene level, we found 353 gene ontology (GO) categories upregulated in EC compared with TX. Our results suggest the existence of mechanisms at two different levels: first boosting both adaptive and innate immune responses, and second promoting active viral replication and halting HIV latency in the Trm cell compartment of ECs as compared with TX patients. These differences in the transcriptional profile of Trm cells could be involved in the lower HIV reservoir observed in ECs compared with TX individuals, although mechanistic studies are needed to confirm this hypothesis. Combining transcriptome analysis and systems biology methods is likely to provide important findings to help us in the design of therapeutic strategies aimed at purging the HIV reservoir. KEY MESSAGES: HIV-elite controllers have the lowest HIV-DNA content in resting memory CD4 T cells. HIV-ECs show a particular transcriptional profile in resting memory CD4 T cells. Molecular mechanisms of enhanced adaptative and innate immune response in HIV-ECs. High viral replication and low viral latency establishment associate to the EC status.

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody-secreting plasma cells. Here, we have addressed the role of activation-induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells, we have identified a novel subset of late-prePB cells (L-prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L-prePB cells and severely impairs transitions between the L-prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state. We thank all the members of the B lymphocyte Biology lab for helpful suggestions, Ana Rodriguez-Ronchel for the elaboration of the graphical abstract, Sonia Mur for technical assistance, Virginia G de Yebenes for critical reading of our manuscript, Julia Merkenschlager, Carlos Torroja, and Enrique Vazquez for their advice on single cell sequencing and analysis, the CNIC Flow Cytometry for assistance on cell analysis and separation and the CNIC Genomics Unit for single cell sequencing. We also thank Sergio Roa, Alicia G Arroyo, Salvador Iborra, and David Sancho for kindly sharing mouse lines and reagents with us. CG-E is supported by a fellowship awarded by La Caixa Espana in ~ 2017 and ASN is an FPI Severo Ochoa fellow (PRE2018-083475). AB, FS-C, and ARR are supported by CNIC. This project was funded by grants from the Spanish Ministerio de Econom ıa, Industria y Competitividad (SAF2016-75511-R), the Spanish Ministerio de Ciencia e Innovaci on (PID2019-106773RB-I00/AEI/10.13039/ 501100011033) and the “la Caixa” Banking Foundation under the project code HR17-00247 to ARR. FS-C is supported by the project RT2018-102084-B-I00 financed by MCIN/AEI/10.13039/5011000110033/ and by FEDER Una Manera de hacer Europa and by Ayuda EQC2021-007294-P financed by MCIN/AEI/ 10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacion (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence, CEX2020-001041-S funded by MICIN/AEI/10.13039/ 501100011033. Sí

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities. This work was supported by Institute of Health Carlos III/ERDF (European Regional Development Fund), Spain [PI16/00409 (DL), PI20/01119 (DL), CP18/00033 (DL), PI15/00227 (MC), CPII16/00037 (SE), and PI18-00286 (SE)], Platform for Proteomics, Genotyping and Cell Lines; PRB3 of ISCIII (PT17/0019/0024); National Science Foundation GACR 18-04393S and the project “Centre of Reconstructive Neuroscience”, registration number CZ.02. 1.01/0.0./0.0/15_003/0000419PI15/00227; Spanish Ministry of Economy and Competitiveness grant BES-2016-076994 (ÁA-L); and Academy of Finland (HS).

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands. This study was supported byIntramural grants from the Severo Ochoa program (IGP-SO), a grant from Fundacio la Marato de TV3 (120/C/2015-20153032), grant SAF2015-65607-R fromMinisterio de Ciencia e Innovacion (MICINN) with co-funding by Fondo Eu-ropeo de Desarrollo Regional (FEDER), RTI2018-095497-B-I00 from MICINN,HR17_00527 from Fundacion La Caixa, and Transatlantic Network of Excel-lence (TNE-18CVD04) from the Leducq Foundation to A.H. I.B. is supportedby fellowship MSCA-IF-EF-748381 and EMBO short-term fellowship 8261.A.R.-P. is supported by a fellowship (BES-2016-076635) and J.A.N.-A. byfellowship SVP-2014-068595 from MICINN. R.O. is supported by ERC startinggrant 759532, Italian Telethon Foundation SR-Tiget grant award F04, ItalianMoH grant GR-201602362156, AIRC MFAG 20247, Cariplo Foundation grant2015-0990, and the EU Infect-ERA 126. C.S. is supported by the SFB 1123,project A07, as well as by the DZHK (German Centre for Cardiovascular Research) and the BMBF (German Ministry of Education and Research) grant81Z0600204. L.G.N. is supported by SIgN core funding from A*STAR. The CNIC is supported by the MICINN and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MICINN award SEV-2015-0505). G.F.-C. issupported by the Spanish Ministerio de Ciencia e Innovacio ́n (grantPID2019-110895RB-100) and Junta de Comunidades de Castilla-La Mancha(grant SBPLY/19/180501/000211). C.R. received funding from the BoehingerIngelheim Foundation (consortium grant ‘‘Novel and Neglected CardiovascularRisk Factors’’) and German Federal Ministry of Education and Research(BMBF 01EO1503) and is a Fellow of the Gutenberg Research College (GFK)at the Johannes Gutenberg-University Mainz Sí

Objectives Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MO) or M-CSF (M-MO), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively. Methods The transcriptomic profile of both human macrophage subtypes exposed to 50 nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints. Results MTX exclusively modulated gene expression in proinflammatory GM-MO, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS + GM-MO are susceptible to MTX, whereas anti-inflammatory TS low/− M-MO and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS + GM-MO. Importantly, TS and p53 were found to be expressed by CD163 + /TNFα + GM-CSF-polarised macrophages from RA joints but not from normal synovium. Conclusions Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro.

Álvarez-Prado et al. report a detailed map of AID-induced off-target mutations and identify molecular features that predict gene mutability. They identify a novel AID hotspot and demonstrate that base excision and mismatch repair back up each other to repair most AID deamination events., Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

Endoglin is an auxiliary cell surface receptor for TGF-β family members. Two different alternatively spliced isoforms, long (L)-endoglin and short (S)-endoglin, have been reported. S-endoglin and L-endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively. A critical role for endoglin in vascular development has primarily been studied in endothelial cells. In addition, endoglin expression is upregulated during monocyte-to-macrophage differentiation; however, little is known about its role in this myeloid context. To investigate the function of endoglin in monocytes, stable transfectants expressing the two endoglin isoforms in the promonocytic human cell line U937 were generated. The differential gene expression fingerprinting of these endoglin transfectants using DNA microarrays and further bioinformatics analysis showed a clear alteration in essential biological functions, mainly those related to "Cellular Movement", including cell adhesion and transmigration. Interestingly, these cellular functions are highly dependent on adhesion molecules, including integrins α1 (CD49a, ITGA1 gene), αL (CD11a, ITGAL gene), αM (CD11b, ITGAM gene) and β2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF-β superfamily member involved in macrophage polarization, was distinctly affected in each endoglin transfectant, and may contribute to the regulated expression of integrins. These data were confirmed by quantitative PCR, flow cytometry and functional tests. Taken together, these results provide new insight into endoglin function in monocytes.

10 pages, 2 figures, 5 tables., CONTEXT: The polycystic ovary syndrome (PCOS) is frequently associated with visceral obesity, suggesting that omental adipose tissue might play an important role in the pathogenesis of the syndrome. OBJECTIVE: The objective was to study the expression profiles of omental fat biopsy samples obtained from morbidly obese women with or without PCOS at the time of bariatric surgery. DESIGN: This was a case-control study. SETTINGS: We conducted the study in an academic hospital. PATIENTS: Eight PCOS patients and seven nonhyperandrogenic women submitted to bariatric surgery because of morbid obesity. INTERVENTIONS: Biopsy samples of omental fat were obtained during bariatric surgery. MAIN OUTCOME MEASURE: The main outcome measure was high-density oligonucleotide arrays. RESULTS: After statistical analysis, we identified changes in the expression patterns of 63 genes between PCOS and control samples. Gene classification was assessed through data mining of Gene Ontology annotations and cluster analysis of dysregulated genes between both groups. These methods highlighted abnormal expression of genes encoding certain components of several biological pathways related to insulin signaling and Wnt signaling, oxidative stress, inflammation, immune function, and lipid metabolism, as well as other genes previously related to PCOS or to the metabolic syndrome. CONCLUSION: The differences in the gene expression profiles in visceral adipose tissue of PCOS patients compared with nonhyperandrogenic women involve multiple genes related to several biological pathways, suggesting that the involvement of abdominal obesity in the pathogenesis of PCOS is more ample than previously thought and is not restricted to the induction of insulin resistance., This work was supported by PI020578, PI020741, PI050341, PI050551, RCMN C03/08, and RGDM 03/212 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, and Grants 08.6/0021/2003 and GR/SAL/0137/2004 from the Consejería de Educación y Cultura, Comunidad de Madrid, Spain.

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized antiphospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R- T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells., This work was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación [grant number SAF2014-52048-R (to A.V.)]; the Consejería de Educación de la Comunidad de Madrid [grant number S2011/BMD-2349 (to A.V.)]; the CSIC program i-COOP+2014 [grant number COOPA20053 (to A.V.)] and the European Commission [grant number PITN-GA-2011-289033 (to A.V.)]; the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program [grant number PITN-GA-2011-289033 (to S.R.S.)]; the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela [grant number CDCH-UCV 03-00-6057-2005 (to V.S.)]; and [grant number PG-03-8728-2013 (to G.B.)]; and the Fondo Nacional de Ciencia, Tecnología e Innovación [grant number P-2011000884 (to G.B.)].

Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromsomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

Aging is a complex process. It consists of a diverse assortment of seemingly random manifestations that occur in the individual, the mutual relationship and impact on mortality of which is frequently obscure. We derive a simple equation to model the aging process based on scale invariant and increasing change. The solution to this equation indicates that this change itself, irrespective of its quality, is the cause and not simply the effect of aging. This model establishes loss of homeostasis as a fundamental feature of aging. The model is deterministic, but it supports the stochastic nature of age changes. Paradoxically, this model states that a sufficient augmentation of aging processes results in a lack of aging. Experimental evidence in support of this model is presented that spans the levels of population mortality rates, cellular spatial organization, and gene dysregulation.

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use. This work was supported by grants to AB from the Ministry of Science and Innovation (SAF 2008-02099; PLE2009-0147 and PSE010000-2009-3), the Comunidad Autonoma de Madrid (P-BIO-0306-2006) and the Red de Terapia Celular del Instituto de Salud Carlos III (TerCel); to ES from the Fundacion Mutua Madrilena, the Ministry of Education (Ramon y Cajal program), the Ministry of Health (FIS PI071023); and to ER from Instituto de Salud Carlos III-FEDER (PS09/01093) and Fundacion Salud 2000-Merck Serono. JCE is a predoctoral fellow funded by TerCel. The CNIC is supported by the Ministry of Economy and Competitiveness and the Pro-CNIC Foundation. Sí

Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through beta-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing beta-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling. We are thankful to C. Giron, C. Guerris, and J. Pairada for their excellent technical assistance. We are greatful to Dr. Loreta Medina for her kind help. This work was supported by Instituto de Salud Carlos III (PI080790) to Judit Herreros and from Grups Consolidats of Generalitat de Catalunya (2009SGRC547). Deepshikha Bhardwaj is a predoctoral fellow of Agaur-Generalitat de Catalunya. Sí

One hundred and sixty Drosophila females laid several thousands of embryos during the 14.5 days of the IML-2 spaceflight. The progeny were either recovered frozen (embryos at final stages of development and larvae), or maintained alive developing further until adulthood. All embryos, larvae, pupae and imagoes recovered were normal in morphology and function. Results from earlier experiments were reproduced in IML-2 with a better experimental design. We confirm that in Space there is a stimulation of oogenesis and that development is slightly delayed when compared to that of synchronous parallel ground controls. Nevertheless, it is clear from the accompanying 1 x g flight control centrifuge and from the 1.4 x g ground centrifuge samples, that centrifugation itself can produce similar effects, emphasizing the importance of reevaluating the role of the 1 x g on-flight controls. The results emphasize the apparent paradox that simple cellular model systems in microgravity show alterations in many fundamental processes such as those involved in cell signalling, while development, relying heavily on these key cellular mechanisms can proceed quite normally in the absence of gravity. The effects on development are small and more the consequence of a reaction to the abnormal Space environment in general than a specific effect of microgravity.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Expansion of human stem cells before cell therapy is typically performed at 20% O(2). Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Here, we demonstrate that culture of human mesenchymal stem cells at lower, physiological O(2) concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Our gene expression and bioenergetic data strongly suggest that growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. We propose that this balance is disturbed at 20% O(2), resulting in abnormally increased levels of oxidative stress. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety.

El pdf del artículo es el manuscrito de autor.-- et al., Pigment epithelium-derived factor (PEDF) is a broad-spectrum angiogenesis inhibitor that displays potent antimetastatic activity in multiple tumor types. We have previously shown that PEDF prevents primary tumor growth and metastatic spread of human melanoma in mouse experimental models. Consistent with these observations, PEDF expression is lost at the late stages of melanoma progression, allowing melanoma cells to become angiogenic, migratory, and invasive. PEDF's ability to modify the interplay between the host and tumor tissues strongly supports its use as a therapeutic agent for the treatment of metastatic melanoma. However, transition to the clinic requires a more detailed knowledge of the molecular mechanisms underpinning PEDF's activity. In this study, we describe changes in the gene expression profile of A375 human melanoma cells induced by PEDF overexpression. PEDF modulated diverse categories of genes known to be involved in angiogenesis and migration. It downregulated cytokines such as interleukin-8 and extracellular matrix proteins such as collagen IV, while it upregulated fibronectin. Multiple transcripts previously described as contributing to the acquisition of malignant phenotype by melanoma were also diminished by PEDF overexpression, among which we validated galectin 3 and jagged 1. In addition, PEDF downregulated S100ß and melanoma inhibitory activity, which are widely used in the pathological diagnosis of melanoma. Interestingly, PEDF increased the expression of melanophilin and decreased rab27A, which are relevant targets for melanosome transport; suggesting that PEDF could directly impinge on melanocytic lineage-specific processes. Our study identifies new molecular targets and signaling pathways that may potentially contribute to determine PEDF's ability to restrict the aggressiveness of A375 human melanoma cells., Supported by grants Ministerio de Educacion y Ciencia grant SAF2007-62292 (BJ), Comunidad de Madrid SAL-0311-2006 (BJ). JLO has been supported by a Ministerio de Educacion y Ciencia fellowship and a SAF2007-62292 contract.

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.

Genome integrity requires faithful chromosome duplication. Origins of replication, the genomic sites at which DNA replication initiates, are scattered throughout the genome. Their mapping at a genomic scale in multicellular organisms has been challenging. In this study we profiled origins in Arabidopsis thaliana by high-throughput sequencing of newly synthesized DNA and identified ~1,500 putative origins genome-wide. This was supported by chromatin immunoprecipitation and microarray (ChIP-chip) experiments to identify ORC1- and CDC6-binding sites. We validated origin activity independently by measuring the abundance of nascent DNA strands. The midpoints of most A. thaliana origin regions are preferentially located within the 5′ half of genes, enriched in G+C, histone H2A.Z, H3K4me2, H3K4me3 and H4K5ac, and depleted in H3K4me1 and H3K9me2. Our data help clarify the epigenetic specification of DNA replication origins in A. thaliana and have implications for other eukaryotes.

The ING family of tumor suppressor proteins controls several cellular functions relevant to antitumor protection, such as cell cycle control, apoptosis, senescence, or migration. ING proteins are functionally linked to the p53 pathway, and they participate in transcriptional control via the recognition of histone marks and recruitment of protein complexes with chromatin-modifying activity to specific promoters. Here, we have investigated the global effect of ING1 in gene regulation through genome-wide analysis of expression profiles in primary embryonic fibroblasts deficient for the Ing1 locus. We find that Ing1 has a predominant role as transcriptional repressor in this setting, affecting the expression of genes involved in a variety of cellular functions. Within the subset of genes showing differential expression, we have identified DGCR8, a protein involved in the early steps of microRNA biogenesis. We show that ING1 binds to the DGCR8 promoter and controls its transcription through chromatin regulation. We also find that ING1 and DGCR8 can cooperate in restraining proliferation. In summary, this study reveals a novel connection between ING1 and a regulator of microRNA biogenesis and identifies new links between tumor suppressor proteins and the microRNA machinery. ©2010 AACR., Spanish Ministry of Science and Innovation grant BFU06-10882 (I. Palmero).

We have reported thatin vitroculture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos culturedin vitroin KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared within vivoderived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos culturedin vitroorin vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

The European Soyuz missions have been one of the main routes for conducting scientific experiments onboard the International Space Station, which is currently in the construction phase. A relatively large number of life and physical sciences experiments as well as technology demonstrations have been carried out during these missions. Included among these experiments are the Gene experiment during the Spanish “Cervantes” Soyuz mission and the ICE-1st experiment during the Dutch “Delta” mission. In both experiments, full genome microarray analyses were carried out on RNA extracted from whole animals recovered from the flight. These experiments indicated relatively large scale changes in gene expression levels in response to spaceflight for two popular model systems, Drosophila melanogaster (Gene) and Caenorabditis elegans (ICE-1st). Here we report a comparative analysis of results from these two experiments. Finding orthologous genes between the fruit fly and the nematode was far from straightforward, reducing the number of genes that we could compare to roughly 20% of the full comparative genome. Within this sub-set of the data (2286 genes), only six genes were found to display identical changes between species (decreased) while 1809 genes displayed no change in either species. Future experiments using ground simulation techniques will allow producing a better, more comprehensive picture of the putative set of genes affected in multicellular organisms by changes in gravity and getting a deeper understanding of how animals respond and adapt to spaceflight., The support of the all organizations and people involved in the Cervantes and Delta missions, the Spanish Space Program (Spanish Ministery of Education and Science), the Dutch Space Program, ESA, the CNES and NASA that made possible this work is gratefully acknowledged.

Manuscript version.-- Final version of the publisher available on http://www.springerlink.com/content/5703g0375l066734/?p=1823d8063e69447cbbb58584de4e2425&pi=2, Human exploration of outer space will eventually take place. In preparation for this endeavour, it is important to establish the nature of the biological response to a prolonged exposure to the space environment. In one of the recent Soyuz Missions to serve the International Space Station (ISS), the Spanish Soyuz mission in October 2003, we exposed four groups of Drosophila male imagoes to microgravity during the almost eleven days of the Cervantes mission to study their motility behaviour. The groups were three of young flies and one of mature flies, In previous space experiments, we have shown that when imagoes are exposed to microgravity they markedly change their behaviour by increasing their motility, especially if subjected to these conditions immediately after hatching. The constraints of the current Soyuz flights made it impossible to study the early post-hatching period. A low temperature cold transport was incorporated as a possible way out of this constraint. It turned out that on top of the space flight effects, the cold treatment by itself, modifies the motility behaviour of the flies. Although the four groups increased their motility, the young flies did it in a much lower extent than the mature flies that had not been exposed to the low temperature during transportation. Nevertheless, the flies flown in the ISS are still more active than the parallel ground controls. As a consequence of the lower motility stimulation in this experiment, a likely consequence of the cold transport step, no effects on the life spans of the flown flies were detected. Together with previous results, this study confirms that high levels of motility behaviour are necessary to produce significant decreases in fly longevity.

6 p.-3 fig., In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae replication forks progressing against transcription stall at a polar replication fork barrier (RFB) located close to and downstream of the 35S transcription unit. Forks blocked at this barrier are potentially recombinogenic. Plasmids bearing the RFB sequence in its active orientation integrated into the chromosomal rDNA in sir2 mutant cells but not in wild-type cells, indicating that the histone deacetylase silencing protein Sir2 (Sir2p), which also modulates the aging process in yeast, suppresses the recombination competence of forks blocked at the rDNA RFB. Orientation of the RFB sequence in its inactive course or its abolition by FOB1 deletion avoided plasmid integration in sir2 mutant cells, indicating that stalling of the forks in the plasmid context was required for recombination to take place. Altogether these results strongly suggest that one of the functions of Sir2p is to modulate access of the recombination machinery to the forks stalled at the rDNA RFB., This work was partially supported by grants PGC PB98-048 from the Spanish Comisión Interministerial de Ciencia y Tecnología, SAF2001-1740 from the Spanish Ministerio de Ciencia y Tecnología and 08.5/0057/2001.1 and 08.1/0067/2001.1 from the Comunidad Autónoma de Madrid.

The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete. Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing. We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity. Deletion of the histone deacetylase gene RPD3 extended life-span. Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3. Similarly, the effect of the rpd3Delta on life-span did not depend on an intact Sir silencing complex. In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging. Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3. The hda1Delta sir3Delta resulted in an increase in silencing, but only at the rDNA locus. Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Delta single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant. Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population. The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span. Our results are consistent with the loss of rDNA silencing leading to aging in yeast. The functions of RPD3 and HDA1 do not overlap entirely. RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process.

While experiments carried out in Space with isolated cells have shown that eucaryotic cells are able to sense and respond to the absence of gravity by modifying their reactions, experiments in which more complex processes have been investigated, such as Biological Systems undergoing development under Microgravity. have been surprisingly unaffected by the space environment. This can be considered a curious result since all organisms are evolutionarily adapted to the current level of the gravity force in our planet and should eventually change if this parameter will vary in a permanent manner. In fact, the small effects of the modifications in gravity on development in short term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical processes such as diffusion-controled auto-organizative reactions in currently developing biological systems. An apparent exception to this lack of effect is experiments where brine shrimp dormant gastrulae directly exposed to the space environment accumulate developmental defects as a consequence of cosmic irradiation. In this article we discuss the idea that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces such as gravity were involved in the evolutionary organization of these primitive organisms. As evolution proceed, these early mechanisms may have been obscured and/or made redundant by the appearance of new internal, environment-independent biological regulatory mechanisms. On the other hand, behavioral responses that may be important, for example, in setting the life-spans of organisms may still be more readily susceptible to manipulation by external cues as experiments carried out by our group in Space and on the ground with Drosophila melanogaster indicate., The financial support of the Spanish National Space Program (ESP97-1775) and of ESA is gratefully acknowledged.

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

Growth factor-stimulated cell proliferation is preceded by a transient increase in the cytoplasmic Ca" concentration,' and calmodulin, an intracellular calcium receptor protein,' appears to intervene in the modulation of this process.3 Calmodulin binds to nuclear proteins and could therefore affect their functions! However, calmodulin does not control cell proliferation by acting exclusively at the nuclear level. Several lines of evidence obtained in our laboratory indicate that the epidermal growth factor receptor (EGFR) is a calmodulin-binding protein and that calmodulin acts as a modulator of its intrinsic tyrosine kinase activity.'.' We have demonstrated that the EGFR can be isolated from solubilized rat liver plasma membranes by calmodulin-affinity chromatography."' Binding of the EGFR to calmodulin-agarose occurs in the presence of Ca'", and its elution is achieved upon addition of EGTA. The preparation obtained contains a set of calmodulinbinding proteins associated with the plasma membrane. However, the major autophosphorylated protein in this preparation corresponds to the EGFR. This autophosphorylation is stimulated by epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) with essentially the same effi~iency.~ Furthermore, the isolated receptor presents both EGFand TGFa-stimulated tyrosine kinase activity towards the exogenous substrate PoIY-L-(GIu : Tyr).'.' The identity of the isolated EGFR

Now known as FEBS Journal: Open Access content older than 1 year., Plasma-membrane-associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(L-lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly(L-lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma-membrane-associated calmodulin was also phosphorylated by plasma-membrane-associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti-calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane-associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(L-lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post-translational modification could have a physiological role in this location., This work was supported by Grants to A. V. from the Comisión lnterministerial de Ciencia y Tecnología (SAF392/93), from the Conejería de Educucición de la Comunidad de Madrid (AE16/94), and from the Direccición General de Investigaciones Científicas y Técnicas (PR94-343) (Spain), and Grant to D. B. S. from the National Institutes of Health (DK43682) (USA). A. B. is the recipient of a predoctoral fellowship from the Departamento de Educación, Universidades e Investigación del Gobierno Vasco (Spain). M. S. is the recipient of a postdoctoral fellowship from the Consejo Superior de Investigaciones Científicas (Spain). J. L. J. is the recipient of a postdoctoral fellowship (DK09062) from the National Institutes of Health (USA).

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin- binding β-galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the α/β-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [125I]EGF to its receptor with similar efficiency., This work was supported in part by grants (toA. V.) from the Comisión Interministerial de Ciencia yTecnología (SAF392/93), and from the Consejería de Educación de la Comunidad de Madrid (366/92) Spain, grants (to H.-J. G.) from the Dr.-M.-Scheel-Stiftung for Krebsforschung, Germany and the Acciones Integradas (H-A 106) between Germany and Spain (to H.-J. G. and A. V.). A.B. is the recipient of a predoctoral fellowship from the Departamento de Educación del Gobierno Vasco, and S.K. is a visiting predoctoral student from the University of Hannover, Germany.

8 pages, 9 figures.-- Now known as FEBS Journal: Open Access content older than 1 year., An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3–8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 32P/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used., This work was supported in part by Research Grants to A. V. from the Comisión Interministerial de Ciencia y Tecnología (SAF392/93), and from the Secretaria de Educación de la Comunidad de Madrid (366/92). A. B. is the recipient of a predoctoral fellowship from the Departamento de Educación, Universidades e Investigación del Gobierno Vasco, 0. H.-P. is the recipient of a predoctoral fellowships from the Cabildo Insular de Gran Canaria, Fundacion Universitaria de Las Palmas, and from the Funducidn Científica de la Asociación Española Contra el Cancer, and M. T. M.-P. is the recepient of an undergraduate traineeship from the Consejo Superior de Investigaciones Cientipcas. Work at D. B. S. lab was supported by Research Grant DK43682 from the National Institutes of Health. USA.

One of the most interesting approaches in the study of lectins is the recognition of defined biochemical pathways and specific cellular responses modulated by these glycoconjugate-binding proteins. In this context, the transduction of signals from lectin receptors, located in the plasma membrane, to specific intracellular target systems is an integral part of these mechanisms and deserves special attention. Biosignaling is, certainly, crucial to understand the mode of action of lectins, a particularly important subject of study in normal and tumor cells when lectins from plant and animal origins are used (Olden and Parent 1987; Gabius 1991).

Background Transcriptional profiling of prostate cancer (PC) has unveiled new markers of neoplasia and allowed insights into mechanisms underlying this disease. Genomewide analyses have also identified new chromosomal abnormalities associated with PC. The combination of both classes of data for the same sample cohort might provide better criteria for identifying relevant factors involved in neoplasia. Here we describe transcriptional signatures identifying distinct normal and tumoral prostate tissue compartments, and the inference and demonstration of a new, highly recurrent copy number gain on chromosome 17q25.3. Methods We have applied transcriptional profiling to tumoral and non-tumoral prostate samples with relatively homogeneous epithelial representations as well as pure stromal tissue from peripheral prostate and cultured cell lines, followed by quantitative RT-PCR validations and immunohistochemical analysis. In addition, we have performed in silico colocalization analysis of co-regulated genes and validation by fluorescent in situ hybridization (FISH). Results The transcriptomic analysis has allowed us to identify signatures corresponding to non-tumoral luminal and tumoral epithelium, basal epithelial cells, and prostate stromal tissue. In addition, in silico analysis of co-regulated expression of physically linked genes has allowed us to predict the occurrence of a copy number gain at chromosomal region 17q25.3. This computational inference was validated by fluorescent in situ hybridization, which showed gains in this region in over 65% of primary and metastatic tumoral samples. Conclusion Our approach permits to directly link gene copy number variations with transcript co-regulation in association with neoplastic states. Therefore, transcriptomic studies of carefully selected samples can unveil new diagnostic markers and transcriptional signatures highly specific of PC, and lead to the discovery of novel genomic abnormalities that may provide additional insights into the causes and mechanisms of prostate cancer.

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca2+-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-α stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration- dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF- stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+., This research was supported in part by Grant PB89/0079 from the Dirección General de Investigación Científica y Técnica and Grant C174/90 from the Consejeria de Educación de la Comunidad de Madrid (to A. V.).