Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy.

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.

Identification of disease-relevant genes for molecularly-targeted drug discovery.

The current paradigm for cancer therapy is undergoing a change from non-specific cytotoxic agents to more specific approaches based on unique molecular features of cancer cells. The identification and validation of disease relevant targets are crucial for the development of molecularly targeted anticancer therapies. Advances in our understanding of the molecular basis of cancer together with novel approaches to interfere with signal transduction pathways have opened new horizons for anticancer target discovery. In particular, the image-based large scale analysis of cellular phenotypes that arise from genetic or chemical perturbations paved the way for the identification and validation of disease relevant molecular targets independent of preconceived notions of mechanistic relationships. In addition, novel and sophisticated techniques of genome manipulation allow for the use of mouse models that faithfully recapitulate critical elements of human cancer for target validation in vivo. We believe that these advances will translate into more and better validated drug targets.

Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis.

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.

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells.

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.

Human TRIB2 is a repressor of FOXO that contributes to the malignant phenotype of melanoma cells.

FOXO transcription factors are evolutionarily conserved proteins that orchestrate gene expression programs known to control a variety of cellular processes such as cell cycle, apoptosis, DNA repair and protection from oxidative stress. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research. In order to discover genes involved in the regulation of FOXO activity, we performed a large-scale RNA-mediated interference (RNAi) screen using cell-based reporter systems that monitor transcriptional activity and subcellular localization of FOXO. We identified genes previously implicated in phosphoinositide 3-kinase/Akt signaling events, which are known to be important for FOXO function. In addition, we discovered a previously unrecognized FOXO-repressor function of TRIB2, the mammalian homolog of the Drosophila gene tribbles. A cancer-profiling array revealed specific overexpression of TRIB2 in malignant melanoma, but not in other types of skin cancer. We provide experimental evidence that TRIB2 transcript levels correlate with the degree of cytoplasmic localization of FOXO3a. Moreover, we show that TRIB2 is important in the maintenance of the oncogenic properties of melanoma cells, as its silencing reduces cell proliferation, colony formation and wound healing. Tumor growth was also substantially reduced upon RNAi-mediated TRIB2 knockdown in an in vivo melanoma xenograft model. Our studies suggest that TRIB2 provides the melanoma cells with growth and survival advantages through the abrogation of FOXO function. Altogether, our results show the potential of large-scale cell-based RNAi screens to identify promising diagnostic markers and therapeutic targets.

Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

Characterization of human gene expression changes after olive oil ingestion: an exploratory approach.

Olive oil consumption is protective against risk factors for cardiovascular and cancer diseases. A nutrigenomic approach was performed to assess whether changes in gene expression could occur in human peripheral blood mononuclear cells after oli ve oil ingestion at postprandial state. Six healthy male volunteers ingested, at fasting state, 50 ml of olive oil. Prior to intervention a 1-week washout period with a controlled diet and sunflower oil as the only source of fat was followed. During the 3 days before and on the intervention day, a very low-phenolic compound diet was followed. At baseline (0 h) and at post-ingestion (6 h), total RNA was isolated and gene expression (29,082 genes) was evaluated by microarray. From microarray data, nutrient-gene interactions were observed in genes related to metabolism, cellular processes, cancer, and atherosclerosis (e.g. USP48 by 2.16; OGT by 1.68-fold change) and associated processes such as inflammation (e.g. AKAP13 by 2.30; IL-10 by 1.66-fold change) and DNA damage (e.g. DCLRE1C by 1.47; POLK by 1.44- fold change). When results obtained by microarray were verified by qRT-PCR in nine genes, full concordance was achieved only in the case of up-regulated genes. Changes were observed at a real-life dose of olive oil, as it is daily consumed in some Mediterranean areas. Our results support the hypothesis that postprandial protective changes related to olive oil consumption could be mediated through gene expression changes.

Incidence of pollinosis in the city of A Coruña: correlation with aerobiological data.

An atmospheric pollen count was carried out in the city of A Coruña during 1999 using two pollen traps located at two different points in the city. A total number of 6979 and 3536 pollen grains, respectively, were identified, the majority during the Spring and Summer. Further, patients living near the pollen traps were selected from among those diagnosed as suffering from respiratory allergies by the Allergy Department of A Coruña's Juan Canalejo Hospital. The patients had at least one positive skin test for some pollen type, had not received immunotherapy in the last year, and were willing to fill in a symptoms booklet during the study period. The results obtained reveal the pollen types that produce the greatest number of skin sensitization cases (Poaceae, Plantago, Chenopodium and Parietaria), with a positive correlation between the atmospheric pollen concentration of such taxa and the frequency of allergy symptoms. This has enabled the setting of pollen values, above which A Coruña's inhabitants are considered to be at risk.

Relationship of clinical and aerobiological pollen data in the north-west of Spain.

BACKGROUND: few studies report clinical and aerobiological pollen data in the north-west of Spain, a region similar to northern and central Europe. Moreover, it is difficult to obtain patients' collaboration in filling out symptom cards. The aim of this study was to establish a relationship between pollen types and clinical data obtained through questionnaire and telephone calls. PATIENTS AND METHODS: from January to December 2000, 24 patients aged 28 10.6 years and allergic to pollens were studied. The seasonal and hourly rhythm of symptoms and their intensity were obtained monthly by telephone calls. Atmospheric pollen was collected over the same period using a Hirst-type volumetric pollen sampler. RESULTS: the most important pollen types recorded were Poaceae, Betula, Parietaria and Plantago. Most patients (83 %) showed symptoms during March and in the period between May and July (99 %), which coincided with the greatest quantity of atmospheric pollen. Fifty-six percent of the patients complained of symptoms during the first hours of the morning, 63 % during the central hours of the day and 22 % at nightfall. In specific sensitizations, symptoms were more evident during the hours of maximum atmospheric levels of their taxa. CONCLUSIONS: the method employed in the present study to obtain information on patients' symptomatology (telephoning their homes once a month) proved useful and revealed a clear relationship between the presence of certain pollens in the atmosphere and the development of symptoms.

Relationship of clinical and aerobiological pollen data in the north-west of Spain

Background: few studies report clinical and aerobiological pollen data in the north-west of Spain, a region similar to northern and central Europe. Moreover, it is difficult to obtain patients' collaboration in filling out symptom cards. The aim of this study was to establish a relationship between pollen types and clinical data obtained through questionnaire and telephone calls. Patients and methods: from January to December 2000, 24 patients aged 28 ± 10.6 years and allergic to pollens were studied. The seasonal and hourly rhythm of symptoms and their intensity were obtained monthly by telephone calls. Atmospheric pollen was collected over the same period using a Hirst-type volumetric pollen sampler. Results: the most important pollen types recorded were Poaceae, Betula, Parietaria and Plantago. Most patients (83 %) showed symptoms during March and in the period between May and July (99 %), which coincided with the greatest quantity of atmospheric pollen. Fifty-six percent of the patients complained of symptoms during the first hours of the morning, 63 % during the central hours of the day and 22 % at nightfall. In specific sensitizations, symptoms were more evident during the hours of maximum atmospheric levels of their taxa. Conclusions: the method employed in the present study to obtain information on patients' symptomatology (telephoning their homes once a month) proved useful and revealed a clear relationship between the presence of certain pollens in the atmosphere and the development of symptoms (AU)

Antecedentes: desde el punto de vista aerobiológico, el noroeste peninsular se aproxima más al Centro y Norte de Europa que al área mediterránea. Son escasos los estudios relacionando síntomas y presencia de polen, en esta zona. Además, es difícil contar con la colaboración de los pacientes para rellenar diarios de síntomas que faciliten esta evaluación. El presente estudio se diseñó para comprobar la posible utilidad de un seguimiento telefónico de los síntomas y su relación con la presencia de pólenes en el ambiente. Pacientes y métodos: se incluyó un total de 24 pacientes adultos, alérgicos a pólenes y cuyo seguimiento se realizó de enero a diciembre del año 2000. Se registró el ritmo estacional y horario de los síntomas obtenido con llamadas telefónicas que mensualmente realizó un investigador. La recogida de pólenes se realizó con un captador volumétrico tipo Hirst situado en el área de influencia a la que los pacientes pertenecían. Resultados: los tipos polínicos presentes con mayor relevancia en la atmósfera fueron Paraceae, Betula, Parietaria y Plantago. La mayoría de los pacientes mostraron síntomas en el mes de marzo (83 %) mientras que en el período de mayo a julio el 99 % de los sujetos se encontraron afectados, coincidiendo con los niveles más elevados de pólenes. Respecto a las variaciones horarias, el 56 % de los pacientes se encontraron peor en las primeras horas de la mañana, el 63 % en las horas centrales del día y tan sólo un 22 %, al anochecer, todo ello en directa relación con la presencia de los distintos tipos polínicos. Conclusiones: el método empleado en el presente estudio ­llamadas telefónicas con periodicidad mensual­ parece útil en el seguimiento de los síntomas, pudiendo detectarse una relación con la presencia de los distintos tipos polínicos en la atmósfera (AU)

Parietaria pollinosis in an Atlantic area: clinical and palynological data.

BACKGROUND: Parietaria pollen is considered as one of the most common causes of allergic respiratory symptoms in the Mediterranean area but its presence is limited in the Atlantic area. Some leading patients from Muros, a small town on the Spanish Atlantic coast, complaining of nearly all year round respiratory symptoms happened to be allergic to Parietaria pollen. AIM OF THE STUDY: To evaluate the prevalence of Parietaria sensitization among patients from this Atlantic town, and its correlation with aerobiological data (concentration of Urticaceae pollen). PATIENTS AND METHODS: Eighty-nine patients suffering from rhinoconjunctivitis and/or asthma from the area of Muros between January 1998 and January 1999 were included. Skin prick tests and serum-specific IgE (CAP Pharmacia) to Parietaria judaica and a battery of perennial or seasonal allergens were performed. Information about the seasonal and hourly rhythm of symptoms was obtained in each patient sensitized to Parietaria pollen. Atmospheric pollen was collected, using a Hirst-type volumetric pollen sampler, during 1998. RESULTS: Parietaria allergy was detected in 22 patients (25%) and represented the second most important aeroallergen after mites and along with grass pollen. The total atmospheric pollen recorded in Muros during the study period was 27,515 pollen grains, Urticaceae being the most important one (18,554 grains, 67% of the total). The proportion of Urticaceae pollen found in Muros was the highest among all samplers belonging to the Spanish Aerobiology Network. Maximum values of Urticaceae pollen were recorded during May and June. Intradiurnal variation of pollen counts showed maximum values between 11 a.m. and 1 p.m. A parallelism was observed between the rate of symptomatic patients and Parietaria type grain pollen count. CONCLUSION: The prevalence of Parietaria pollen sensitization seems to be very important in this Atlantic area. The presence of very high levels of this pollen in its atmosphere explains this fact. Such sensitization should be taken into account concerning specific diagnostic tests.

Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.

Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction.

We report an improved subtractive cDNA cloning procedure, named "directional tag PCR subtraction," for isolating clones of mRNAs enriched in a target tissue compared to a second tissue, the driver. In this method, the target and driver are prepared from directional cDNA libraries constructed in different vectors, and the target cDNA contains tag sequences at both its 5' and 3' ends for PCR amplification. This method avoids several limitations of previous subtraction procedures, and was demonstrated to be technically easy and efficient. Using the directional tag PCR subtraction and improved screening procedures, cDNA clones corresponding to mRNAs expressed in the striatum but not in the cerebellum of the rat brain were efficiently isolated, including mRNAs encoding calmodulin-dependent phosphodiesterase, a transcriptional regulatory protein, and several previously uncharacterized species. Our data suggest that approximately 1% of the striatal polyA+ RNA mass potentially encoding more than 300 distinct proteins corresponds to RNA species reduced in concentration or absent from the cerebellum, of which about one-third are expressed prominently only in the striatum. This unexpected finding suggests that the striatum has a unique biochemical character within the brain, and that characterization of these mRNAs will be important for understanding the biochemical basis of striatal function.

Characterisation of mutant alleles of the cell division protein FtsA, a regulator and structural component of the Escherichia coli septator.

Two alleles of ftsA, a gene that encodes an essential cell division protein in Escherichia coli, have-been mapped at the nucleotide level. The mutations are located inside domains that are conserved in an ATP-binding protein family. The ftsA2 mutation lies in the adenine-binding domain, and the ftsA3 in the ribose-binding domain. The defect in ampicillin binding to PBP3 described for allele ftsA3 is allele-specific. This supports the hypothesis of the existence of different domains in FtsA having different functions.

Primary structure of mouse secretogranin III and its absence from deficient mice.

The rat brain- and pituitary-specific 1B1075 mRNA encodes a chromogranin/secretogranin-like protein, called secretogranin III (SgIII), that is a component of intracellular dense core vesicles. In order to study the function of this gene product in a mouse model system, we have isolated the murine homolog of the rat 1B1075 mRNA. This mRNA contains 2163 bp encoding a putative protein of 421 amino acids. Cleavage of the strong putative signal sequence would yield a mature protein of 51 kDa. The sequence of the encoded murine protein preserves the structural features that suggest SgIII is a member of the granin family, and allowed us to recognize and correct errors in our published rat sequence.

An amino-proximal domain required for the localization of FtsQ in the cytoplasmic membrane, and for its biological function in Escherichia coli.

The location of FtsQ, an Escherichia coli protein essential for cell division, is, under physiological conditions, in the cytoplasmic membrane facing towards the periplasmic space. An amino-proximal hydrophobic domain is required for FtsQ to reach its location and for its activity in the cell. Overexpression of modified forms of FtsQ is deleterious for the cell.

On the chronology and topography of bacterial cell division.

Gene products that play a role in the formation of cell septum should be expected to be endowed with a set of specific properties. In principle, septal proteins should be located at the cell envelope. The expression of division genes should ensure the synthesis of septal proteins at levels commensurate with the needs of cell division at different rates of cell duplication. We have results indicating that some fts genes located within the 2.5-min cluster in the Escherichia coli chromosome conform to these predictions.

The native form of FtsA, a septal protein of Escherichia coli, is located in the cytoplasmic membrane.

Antisera able to recognize FtsA, one of the septal proteins of Escherichia coli, have been obtained and used to show that native FtsA, when expressed at levels ranging from physiological to induced from lambda pR, is located in the inner membrane. Experiments of trypsin accessibility to FtsA in membranes, spheroplasts, and vesicles indicated that FtsA is located such that it faces the cytoplasm. This location is consistent with current knowledge about the participation of FtsA in a molecular complex active in cell division called septator.

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.