Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.</p

Splicing of the platelet‐derived‐growth‐factor A‐chain mRNA in human malignant mesothelioma cell lines and regulation of its expression

Platelet‐derived‐growth‐factor (PDGF) A‐chain transcripts differing in the presence or absence of an alternative exon‐derived sequence have been described. In some publications, the presence of PDGF A‐chain transcripts with this exon‐6‐derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse‐transcription polymerase‐chain‐reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A‐chain transcript without the exon‐6‐derived sequence. This sequence encodes a cell‐retention signal, which means that the PDGF A‐chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A‐chain protein might be involved in autocrine growth stimulation via PDGF α receptors. However, human malignant mesothelioma cell lines only possess PDGF β receptors. If this also holds true in vivo, the PDGF A‐chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A‐chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern‐blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A‐chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A‐chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein‐synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A‐chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run‐off analysis showed that nuclear PDGF A‐chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A‐chain gene in human malignant mesothelioma cell lines.</p

Physiological effects of water flow induced swimming exercise in seabream Sparus aurata

A longer on-land rearing period of Gilthead seabream Sparus aurata before transfer to sea-cages would allow the farmer to benefit from exercise-enhanced growth, resilience, and robustness as induced by increasing water flow in the tanks. In this study, the physiological effects of flow-conditioning were investigated by subjecting large groups of experimental fish to minimal flow or to flow regimes inducing swimming exercise at 1 or 2 body length (BL) s−1 for a period of 8 months (February–October) in 1,500 L tanks. Fish representing the three treatment groups were then used for: (1) a stress challenge netting test and plasma cortisol measurement (baseline, peaking, and recovery levels), (2) blood plasma measurements of glucose, triglycerides, lactate, cholesterol, growth hormone (GH), and insulin-like growth factor 1 (IGF1), and (3) heart and muscle gene expression of the GH and IGF1 receptors and the muscle transcriptome by deep RNA sequencing (RNAseq). Fish size after 8 months of flow conditioning was 92 ± 27 g body weight (BW) for fish under minimal flow, 106 ± 24 g BW (+15%) at 1 BL s−1, and 125 ± 27 g BW (+36%) at 2 BL s−1. Flow conditioning at 1 BL s−1 provided optimal conditions for growth and uniformity, but also stress (lowest baseline plasma cortisol), robustness (higher condition factor and larger hearts), and energy mobilization (increased plasma glucose). Although flow enhanced growth linearly with swimming speed, also the percentage of lordotic fish increased with exercise, particularly high for swimming at 2 BL s−1. The absence of important differences in plasma GH and IGF1, and expression levels of their receptors in heart and white skeletal muscle, indicated that other factors may be involved in growth enhancement. RNAseq of the white skeletal muscle showed upregulated expression of genes involved in muscle contraction, muscle development and its molecular regulation, and immune genes that may play a role in the muscle repair mechanism. An exercise regime of swimming at 1 BL s−1 can be considered as optimal for farming robust seabream although the increase of skeletal deformities should be avoided.info:eu-repo/semantics/publishedVersio

Physiological effects of water flow induced swimming exercise in seabream Sparus aurata

A longer on-land rearing period of Gilthead seabream Sparus aurata before transfer to sea-cages would allow the farmer to benefit from exercise-enhanced growth, resilience, and robustness as induced by increasing water flow in the tanks. In this study, the physiological effects of flow-conditioning were investigated by subjecting large groups of experimental fish to minimal flow or to flow regimes inducing swimming exercise at 1 or 2 body length (BL) s−1 for a period of 8 months (February–October) in 1,500 L tanks. Fish representing the three treatment groups were then used for: (1) a stress challenge netting test and plasma cortisol measurement (baseline, peaking, and recovery levels), (2) blood plasma measurements of glucose, triglycerides, lactate, cholesterol, growth hormone (GH), and insulin-like growth factor 1 (IGF1), and (3) heart and muscle gene expression of the GH and IGF1 receptors and the muscle transcriptome by deep RNA sequencing (RNAseq). Fish size after 8 months of flow conditioning was 92 ± 27 g body weight (BW) for fish under minimal flow, 106 ± 24 g BW (+15%) at 1 BL s−1, and 125 ± 27 g BW (+36%) at 2 BL s−1. Flow conditioning at 1 BL s−1 provided optimal conditions for growth and uniformity, but also stress (lowest baseline plasma cortisol), robustness (higher condition factor and larger hearts), and energy mobilization (increased plasma glucose). Although flow enhanced growth linearly with swimming speed, also the percentage of lordotic fish increased with exercise, particularly high for swimming at 2 BL s−1. The absence of important differences in plasma GH and IGF1, and expression levels of their receptors in heart and white skeletal muscle, indicated that other factors may be involved in growth enhancement. RNAseq of the white skeletal muscle showed upregulated expression of genes involved in muscle contraction, muscle development and its molecular regulation, and immune genes that may play a role in the muscle repair mechanism. An exercise regime of swimming at 1 BL s−1 can be considered as optimal for farming robust seabream although the increase of skeletal deformities should be avoided.info:eu-repo/semantics/publishedVersio

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae)

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2\u2032,4\u2032-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC\uaeCRL-2536TM) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2\u2032,4\u2032-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1mM), Piperine and Plumbagin (0.25mM), while 2\u2032,4\u2032-Dihydroxychalcone considerably slowed downmycelial growth for 24 h at a concentration of 0.1mM. Therefore, this research allowed to identify two compounds, Tomatine and 2\u2032,4\u2032-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers

Manipulating Heat Shock Factor-1 in Xenopus Tadpoles: Neuronal Tissues Are Refractory to Exogenous Expression

Contains fulltext : 83587.pdf (publisher's version ) (Open Access)10 p

Temperature modulates testis steroidogenesis in European eel

[EN] This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10 degrees C (first 4 weeks), 15 degrees C (next 3 weeks) and 20 degrees C (last 6 weeks); T15: 15 degrees C (first 4 weeks) and 20 degrees C (last 9 weeks); and T20: constant 20 degrees C for the duration of the experiment. At 10 degrees C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was >= 15 degrees C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11 beta HSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2).Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was >= 15 degrees C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration. (C) 2016 Elsevier Inc. All rights reserved.This study was funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no245257 (PRO-EEL), VLC/CAMPUS Program (SP.20140630) and by the MINECO (REPRO-TEMP; AGL2013-41646-R). V.G. and I.M. had predoctoral grants from MINECO (BES-2009-020310) and Generalitat Valenciana, respectively. M.C.V. and M.M. have predoctoral grants from UPV (2011-S2-02-6521) and Generalitat Valenciana (Programa Grisolia), respectively. D.S.P. was supported by MICINN and UPV (PTA2011-4948-I) and was granted with a Short-Term Scientific Mission to make the steroids analyses in Tromso by COST Office (COST Action FA1205: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry; AQUAGAMETE).Peñaranda, D.; Morini, M.; Tveiten, H.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Dirks, R.; Van Den Thillart, GE.... (2016). Temperature modulates testis steroidogenesis in European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 197:58-67. https://doi.org/10.1016/j.cbpa.2016.03.012S586719

Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates

Since its discovery in mammals as a key-hormone in reproduction and metabolism, leptin has been identified in an increasing number of tetrapods and teleosts. Tetrapods possess only one leptin gene, while most teleosts possess two leptin genes, as a result of the teleost third whole genome duplication event (3R). Leptin acts through a specific receptor (LEPR). In the European and Japanese eels, we identified two leptin genes, and for the first time in vertebrates, two LEPR genes. Synteny analyses indicated that eel LEPRa and LEPRb result from teleost 3R. LEPRb seems to have been lost in the teleost lineage shortly after the elopomorph divergence. Quantitative PCRs revealed a wide distribution of leptins and LEPRs in the European eel, including tissues involved in metabolism and reproduction. Noticeably, leptin1 was expressed in fat tissue, while leptin2 in the liver, reflecting subfunctionalization. Four-month fasting had no impact on the expression of leptins and LEPRs in control European eels. This might be related to the remarkable adaptation of silver eel metabolism to long-term fasting throughout the reproductive oceanic migration. In contrast, sexual maturation induced differential increases in the expression of leptins and LEPRs in the BPG-liver axis. Leptin2 was strikingly upregulated in the liver, the central organ of the reproductive metabolic challenge in teleosts. LEPRs were differentially regulated during sexual maturation, which may have contributed to the conservation of the duplicated LEPRs in this species. This suggests an ancient and positive role of the leptin system in the vertebrate reproductive function. This study brings new insights on the evolutionary history of the leptin system in vertebrates. Among extant vertebrates, the eel represents a unique case of duplicated leptins and leptin receptors as a result of 3R

Cortisol Acting Through the Glucocorticoid Receptor Is Not Involved in Exercise-Enhanced Growth, But Does Affect the White Skeletal Muscle Transcriptome in Zebrafish (Danio rerio)

Forced sustained swimming exercise at optimal speed enhances growth in many fish species, particularly through hypertrophy of the white skeletal muscle. The exact mechanism of this effect has not been resolved yet. To explore the role of cortisol, we first subjected wild-type zebrafish to an exercise protocol validated for exercise-enhanced growth, and showed that exercised zebrafish, which indeed showed enhanced growth, had higher cortisol levels than the non-exercised controls. A central role was therefore hypothesized for the steroid hormone cortisol acting through the Glucocorticoid receptor (Gr). Second, we subjected wild-type zebrafish and zebrafish with a mutant Gr to exercise at optimal, suboptimal, and super-optimal speeds and compared them with non-exercised controls. Exercised zebrafish showed growth enhancement at all speeds, with highest growth at optimal speeds. In the Gr mutant fish, exercise resulted in growth enhancement similar to wild-type zebrafish, indicating that cortisol signaling through Gr cannot be considered as a main determinant of exercise-enhanced growth. Finally, the transcriptome of white skeletal muscle tissue was analyzed by RNA sequencing. The results of this analysis showed that in the muscle tissue of Gr mutant fish a lower number of genes is regulated by exercise than in wild-type fish (183 vs. 351). A cluster of 36 genes was regulated by exercise in both wild-type and mutant fish, and in this cluster genes involved in transcriptional regulation and protein ubiquitination were overrepresented. Because these two processes appear to be regulated in both wild type and mutant fish, which both display exercise-enhanced growth, we suggest that they play an important role in the growth of muscles upon exercise