Thyrotropin-dependent proliferation of in vitro rat thyroid cell systems

18 pages, 2 figures, 1 table.-- Review.This review is focused on the most recent knowledge on growth control of rat thyroid cell lines. We analyzed the effect of mitogenic as well as inhibitory agents, but mainly the proliferative effect elicited by thyrotropin (TSH). The classic cAMP-dependent protein kinase (PKA) signal transduction pathway involved in TSH-mediated cell growth is analyzed exhaustively. We have also reviewed new concepts about the participation of other effectors such as small GTPases and phosphatidyl inositol-3-kinase (PI3-K) and the new data about the existence of a cAMP-dependent but PKA-independent pathway. Finally, we give information about TSH induction of cell cycle-related genes, such as G1 cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors.We are grateful to Dirección General de Investigación Científica y Tecnológica (Grant PM97±0065), Comunidad Autónoma de Madrid (Grants 08.1/0025.1/97 and 1/99) and Fundación Salud 2000 (Spain) for supporting our work. DLM has a Fellowship from the Ministerio de Educación y Cultura (Spain)Peer reviewe

A reverse-engineering approach to dissect post-translational modulators of transcription factor's activity from transcriptional data.

BACKGROUND: Transcription factors (TFs) act downstream of the major signalling pathways functioning as master regulators of cell fate. Their activity is tightly regulated at the transcriptional, post-transcriptional and post-translational level. Proteins modifying TF activity are not easily identified by experimental high-throughput methods. RESULTS: We developed a computational strategy, called Differential Multi-Information (DMI), to infer post-translational modulators of a transcription factor from a compendium of gene expression profiles (GEPs). DMI is built on the hypothesis that the modulator of a TF (i.e. kinase/phosphatases), when expressed in the cell, will cause the TF target genes to be co-expressed. On the contrary, when the modulator is not expressed, the TF will be inactive resulting in a loss of co-regulation across its target genes. DMI detects the occurrence of changes in target gene co-regulation for each candidate modulator, using a measure called Multi-Information. We validated the DMI approach on a compendium of 5,372 GEPs showing its predictive ability in correctly identifying kinases regulating the activity of 14 different transcription factors. CONCLUSIONS: DMI can be used in combination with experimental approaches as high-throughput screening to efficiently improve both pathway and target discovery. An on-line web-tool enabling the user to use DMI to identify post-transcriptional modulators of a transcription factor of interest che be found at http://dmi.tigem.it

RhoA activation promotes transformation and loss of thyroid cell differentiation interfering with thyroid transcription factor-1 activity

12 pages, 8 figures, 1 table.Highly specialized cells, the thyrocytes, express a thyroid-specific set of genes for thyroglobulin (Tg), thyroperoxidase, and the transcription factors TTF-1, TTF-2, and Pax-8. The implication of the small GTPase RhoA in TSH-mediated proliferation of FRTL-5 rat thyroid cells has been previously demonstrated. To further analyze RhoA function in thyroid cell proliferation and differentiation patterns, we combined transient and stable transfection assays to express different mutant RhoA forms in FRTL-5 cells. Constitutively active RhoA (FRTL-5-RhoA QL cells) exhibited a fibroblast-like phenotype with organized actin fibers, whereas cells expressing the RhoA negative dominant phenotype (FRTL-5-RhoA N19 cells) present a rounded morphology and lose normal cytoskeletal architecture. In addition, expression of the constitutively active form of RhoA results in TSH-independent proliferation and anchorage-independent growth and induces tumors when inoculated in nude mice. Interestingly, FRTL-5-RhoA QL cells express less Tg and TTF-1 than wild-type FRTL-5 (FRTL-5- vector) or FRTL-5-RhoA N19, suggesting a loss at the differentiation stage. This effect is mediated, at least in part, by a decrease in TTF-1 activity, since transient or stable expression of RhoA QL results in a reduction in the activity of the wild-type Tg promoter as well as an artificial promoter the activation of which depends exclusively on TTF-1. The similarity between RhoA effects and thyroid transformation by Ras suggests that RhoA may act as a downstream effector of Ras; in fact, the dominant negative RhoA N19 abolished the down- regulatory effect of Ras V12 over the Tg promoter. Taken together, these results show for the first time that active RhoA is able to transform FRTL-5 cells and that this effect is coupled to a loss of thyroid differentiation due to impaired TTF-1 activity.This work was supported by DGICYT Grants PM97/0065 and BMC2001-2087, and CAM Grant 08.1/0025/99. D.L.M. and M.R. are recipients of a fellowship from the Spanish Ministerio de Educación y Cultura and Ciencia y Tecnología, respectively.Peer reviewe

Thyroid status modulates T lymphoma growth via cell cycle regulatory proteins and angiogenesis

We have shown in vitro that thyroid hormones (THs) regulate the balance between proliferation and apoptosis of T lymphoma cells. The effects of THs on tumor development have been studied, but the results are still controversial. Herein, we show the modulatory action of thyroid status on the in vivo growth of T lymphoma cells. For this purpose, euthyroid, hypothyroid, and hyperthyroid mice received inoculations of EL4 cells to allow the development of solid tumors. Tumors in the hyperthyroid animals exhibited a higher growth rate, as evidenced by the early appearance of palpable solid tumors and the increased tumor volume. These results are consistent with the rate of cell division determined by staining tumor cells with carboxyfluorescein succinimidyl ester. Additionally, hyperthyroid mice exhibited reduced survival. Hypothyroid mice did not differ significantly from the euthyroid controls with respect to these parameters. Additionally, only tumors from hyperthyroid animals had increased expression levels of proliferating cell nuclear antigen and active caspase 3. Differential expression of cell cycle regulatory proteins was also observed. The levels of cyclins D1 and D3 were augmented in the tumors of the hyperthyroid animals, whereas the cell cycle inhibitors p16/INK4A (CDKN2A) and p27/Kip1 (CDKN1B) and the tumor suppressor p53 (TRP53) were increased in hypothyroid mice. Intratumoral and peritumoral vasculogenesis was increased only in hyperthyroid mice. Therefore, we propose that the thyroid status modulates the in vivo growth of EL4 T lymphoma through the regulation of cyclin, cyclin-dependent kinase inhibitor, and tumor suppressor gene expression, as well as the stimulation of angiogenesis.Fil: Sterle, Helena Andrea. Pontificia Universidad Católica Argentina ; ArgentinaFil: Valli, Eduardo. Pontificia Universidad Católica Argentina ; ArgentinaFil: Cayrol, Maria Florencia. Pontificia Universidad Católica Argentina ; ArgentinaFil: Paulazo, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Martinel Lamas, Diego José. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Díaz Flaqué, María Celeste. Pontificia Universidad Católica Argentina ; ArgentinaFil: Klecha, Alicia Juana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Colombo, L.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cremaschi, Graciela Alicia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Pontificia Universidad Católica Argentina ; ArgentinaFil: Barreiro Arcos, María Laura. Pontificia Universidad Católica Argentina ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Antígenos leucocitarios como genes candidatos para mejorar la respuesta inmune en cerdos

Leukocyte antigens (CD) have functions related to immune response and are of interest as classical candidate genes for health. Polymorphisms (e.g. SNPs) in these genes may be associated with variation in the immune response and consequently in disease response. This approach is being taken in search of susceptibility genes for swine disease. In addition, these genes may vary between populations, especially where specific adaptation to pathogens has occurred, and are of potential interest in characterising pig biodiversity.Los antígenos leucocitarios (CD) tienen funciones relacionadas con la respuesta inmune y son de interés con genes candidatos clásicos para la salud. Los Polimorfismos (ej. SNPs) en estos genes pueden estar asociados con variaciones en la respuesta inmune y consecuentemente con la respuesta a la enfermedad. Este ensayo se está desarrollando en la búsqueda de susceptibilidades genéticas a enfermedades porcinas. Adicionalmente, estos genes pueden variar entre poblaciones, especialmente en la que han ocurrido adaptaciones a patógenos, y suponen un interés potencial para la caracterización de la biodiversidad porcina

Membrane insertion of soluble CLIC1 into active chloride channels is triggered by specific divalent cations

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+ and Zn2+ trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+ leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+ mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+ and Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENT CLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+ and Zn2+ binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition

A 5LCHB Inverter for PV transformerless applications with reduced leakage ground current

Transformerless inverters for photovoltaic systems are widely used as it features low cost, volume, and weight. Thus, in recent years, its study has been of great interest to the research community. In this paper a transformerless cascade multilevel inverter for photovoltaic applications with leakage ground current compensation capability is presented. The proposed solution involves a second-order LC output filter with a particular connection, which is referred to as the DC-link-tied LC output filter. This solution is aimed to deal with the leakage-ground current issue, regardless of the considered PWM strategy. The mathematical model of the system involving such a particular LC output passive filter configuration is presented, out of which, both the differential-mode and the common-mode models are obtained. These models are used to explain the leakage-ground current improvement of the proposed DC-link-tied LC output filter. This hardware solution is evaluated under different modulation schemes to contrast the converter output response and the leakage-ground current performance. Finally, simulation and experimental results are performed using a 1 kW academic prototype to assess the performance of the proposed DC-link-tied LC output filter used in a transformerless inverter application.Peer ReviewedObjectius de Desenvolupament Sostenible::7 - Energia Assequible i No ContaminantObjectius de Desenvolupament Sostenible::11 - Ciutats i Comunitats SosteniblesPostprint (published version

Atlantic bluefin tuna spawn at suboptimal temperatures for their offspring

Life-history traits such as spawning migrations and timing of reproduction are adaptations to specific environmental constraints and seasonal cycles in many organisms’ annual routines. In this study we analyse how offspring fitness constrains spawning phenology in a large migratory apex predator, the Atlantic bluefin tuna. The reproductive schedule of Atlantic bluefin tuna varies between spawning sites, suggesting plasticity to local environ- mental conditions. Generally, temperature is considered to be the main constraint on tuna spawning phenology. We combine evidence from long- term field data, temperature-controlled rearing experiments on eggs and larvae, and a model of egg fitness, and show that Atlantic bluefin tuna do not spawn to optimize egg and larval temperature exposure. The timing of spawning leads to temperature exposure considerably lower than optimal at all spawning grounds across the Atlantic Ocean. The early spawning is constrained by thermal inhibition of egg hatching and larval growth rates, but some other factors must prevent later spawning. Matching offspring with ocean productivity and the prey peak might be an important driver for bluefin tuna spawning phenology. This finding is important for predictions of reproductive timing in future climate warming scenarios for bluefin tuna.Versión del edito

Pelagic habitat and offspring survival in the eastern stock of Atlantic bluefin tuna

In this manuscript, we test how an understanding of geographical variation in larval fitness in relation to temperature and habitat use could be a useful method to improve our understanding of recruitment and develop better indices of annual recruitment. On the basis of the assumption that growth and survival of tuna larvae are influenced by temperature, we have developed a potential larval survival index for Atlantic bluefin tuna (Thunnus thynnus) by combining empirical data from egg and larval rearing experiments with temperature data from hydrodynamic models. The experiments were designed to test the full range of temperature variability that bluefin larvae would experience in the field and provide a mechanistic understanding of the processes driving egg and larval survival. We then developed a biological model using the temperature-related growth expressions and a size-dependent survival function for the larvae. The biological model was applied to a time-series of spatially explicit temperature data for the western Mediterranean from the Strait of Gibraltar to 6 E, which includes the major recognized bluefin tuna eastern stock spawning area, the Balearic Sea. Our results show that areas with high probabilities of larval survival coincide with those that would be considered as optimal based on other data sources (ichthyoplankton surveys, spawning female locations from commercial fisheries data, and adult tracking data). However, evidence of spawning has been found in areas with suboptimal thermal habitats, as predicted by the model, which we discuss regarding sampling effort and salinity fronts. There was a good match between the survival index and recruitment indices from standardized CPUE fisheries data. These results have implications for our understanding of the recruitment process of the eastern stock of Atlantic bluefin tuna, since they suggest that the combined effects of temporal and spatial variability of the environment drive recruitment success, which has important implications for the management of the species.Versión del editor2,27