Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE)

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event

Ultrastructural characterisation of the cell wall of Arabidopsis thaliana transgenic plants

The plant cell wall is a strong fibrillar network that gives each cell its stable shape. It is constituted by a network of cellulose microfibrils embedded in a matrix of polysaccharides, such as xyloglucans. To enlarge, cells selectively loosen this network. Moreover, there is a pectin-rich intercellular material, the middle lamella, cementing together the walls of adjacent plant cells. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a group of enzymes involved in the reorganisation of the cellulose-xyloglucan framework by catalysing cleavage and re-ligation of the xyloglucan chains in the plant cell wall, and are considered cell wall loosening agents. In the laboratory, it has been isolated and characterised a XTH gene, ZmXTH1, from an elongation root cDNA library of maize. To address the cellular function of ZmXTH1, transgenic Arabidopsis thaliana plants over-expressing ZmXTH1 (under the control of the CaMV35S promoter) were generated. The aim of the work performed was therefore the characterisation of these transgenic plants at the ultrastructural level, by transmission electron microscopy (TEM).The detailed cellular phenotype of transgenic plants was investigated by comparing ultra-thin transverse sections of basal stem of 5-weeks old plants of wild type (Col 0) and 35S-ZmXTH1 Arabidopsis plants. Transgenic plants show modifications in the cell walls, particularly a thicker middle lamella layer with respect the wild type plants, supporting the idea that the overexpression of ZmXTH1 could imply a pronounced wall-loosening. In sum, the work carried out reinforces the idea that ZmXTH1 is involved in the cell wall loosening process in maize.  

Implication of Nerve Growth Factor in intestinal mucosal mast cell activity and colonic motor alterations in a model of ovalbumin-induced gut dysfunction in rats

We determined NGF involvement in MMCs and colonic motor alterations in an ovalbumin (OVA)-induced gut dysfunction model in rats. Animals received OVA (6 weeks), with/without simultaneous K252a (TrkA antagonist) treatment. MMCs, rat mast cell protease II (RMCPII) levels and colonic contractility in vitro were assessed. OVA increased MMC density and RMCPII concentration. Spontaneous contractility was similar in both groups and inhibited by K252a. Carbachol responses were increased by OVA in a K252a-independent manner. NO-synthase inhibition increased spontaneous activity in OVA-treated animals in a K252a-dependent manner. These observations support an involvement of NGF in the functional changes observed in this model.

High tidal volume ventilation affects neuronal activation in mechanically ventilated rats

Els malalts crítics presenten sovint seqüeles cognitives a llarg termini, l’aplicació de ventilació mecànica (VM) pot contribuir al seu desenvolupament. El principal objectiu del nostre estudi fou investigar l’efecte de dos patrons de ventilació (volum corrent elevat/baix) en l’activació neuronal (expressió de c-fos) en determinades àrees cerebrals en un model en rates. Després de 3 hores sota VM, es va trobar activació neuronal; la seva intensitat va ser superior al grup de volum corrent elevat, suggerint un efecte iatrogènic de la VM al cervell. Aquests resultats suggereixen que cal aprofundir en l’estudi del crosstalk cervell-pulmó en malalts crítics sota VM.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Background: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.Results: The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.Conclusion: Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.

Traducción comentada de “Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology”

The purpose of this paper is to provide a translation into Spanish of a review articleabout genetically modified organisms (GMOs) entitled “Genetically ModifiedOrganisms (GMOs): Transgenic Crops and Recombinant DNA Technology” publishedby the well-known scientific journal Nature. In a world where English has become thelingua franca when it comes to transferring scientific knowledge and information, itmust be taken into account that not everyone—from scientist to the general public—hasa good enough command of English so that they can feel comfortable enough reading inthis language. Translators are consequently needed resulting from a great demand oftranslation activity into, for example, Spanish. This is the reason why the proposedSpanish translation is followed by a detailed analysis emphasizing the difficulties andproblems that characterize scientific—and also general—translation (i.e. terminology,syntax, semantics, pragmatics, and ideology), for which different approaches as how tosolve them are provided. On the basis of the analysis, it can be concluded thatexperience will be of much help to scientific translators, given that specificterminological knowledge and style requirements must always be born in mind whentranslating in this field. Moreover, this paper is intended to serve as a guide forTranslation students specializing in the field of science and the expectation is to helpthem make the right decisions when it comes to translating. However, it is clear that itcan only be thought of as an introduction that should be completed with further researchand documentation tasks in order to offer a complete reference tool: the ultimatehandbook of scientific translation.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats.

Background and aims: The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with powerful vascular permeability effects. We assessed regional blood flow, vascular leakage, mRNA and tissular expression of VEGF-A and Ang-2 and vascular permeability following VEGF receptor 2 blockade in control and cirrhotic rats to define the vascular territories showing altered vascular permeability in cirrhosis and to determine whether VEGF-A and Ang-2 are involved in this phenomenon. Methods: Arterial blood flow was analysed with the coloured microsphere method. Vascular leakage was measured and visualised with the dye Evan¿s Blue and colloidal carbon techniques, respectively. VEGF-A and Ang-2 expression were determined by real-time polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. The effect on vascular permeability induced by VEGFR2 blockade was assessed by administration of the receptor inhibitor SU11248. Results: Arterial blood flow was increased in the mesentery, pancreas and small intestine but not in the kidney and spleen of cirrhotic rats as compared to controls. Increased vascular leakage was observed in the mesentery and liver, where colloidal carbon spread from microvessels to the adjacent fibrotic tracts. Increased hepatic and mesenteric expression of VEGF-A and Ang-2 was found in cirrhotic rats as compared to controls. Blockade of VEGFR2 markedly reduced hepatic and mesenteric vascular leakage in cirrhotic rats. Conclusions: Enhanced endothelial permeability is restricted to the hepatic and mesenteric vascular beds in cirrhotic rats with ascites and VEGF-A and Ang-2 are key factors in the signalling pathways regulating this dysfunction.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Blood lymphocyte subsets in rats with adjuvant arthritis

To determine the phenotype of peripheral blood lymphocytes during the time-course of adjuvant arthritis (AA) to detect alterations that could be involved in the pathogenesis of the arthritic process. METHODS--Phenotype analysis was performed on days 7, 14, 21, 28, 42, 56 and 70 after arthritis induction using monoclonal antibodies to CD5, CD4 and CD8 subsets, and flow cytometry. The proportion of activated lymphocytes and lymphocytes was also assessed with monoclonal antibodies to IL-2R (CD25), to Ia antigen and by polyclonal antibodies to rat Ig. RESULTS--Adjuvant arthritis produced leukocytosis with neutrophilia. Rats with AA showed a marked increase in the number of both CD4+ and CD8+ cells. The ratio CD4/CD8 decreased because the rise in CD8+ cells was more pronounced than the increase in CD4+ cells. Changes in lymphocyte counts showed two well-defined periods: the first, from day 14 to day 28, during which the inflammation of the joints reached a maximum and changes in lymphocyte subsets were more pronounced, that is, there was a threefold increase in CD8+ lymphocytes over normal counts, and the second, from day 42 to day 70, in which modified parameters improved considerably but remained different from controls. CONCLUSION--Alterations were detected in the phenotype of peripheral blood lymphocytes in AA, which provides an additional marker of disease activity.

Nitric oxide synthase (NOS) inhibition for one week improves renal sodium and water excretion in cirrhotic rats with ascites

Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.

Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.

Bacterial translocation in cirrhotic rats. Its role in the development of spontaneous bacterial peritonitis

Bacterial translocation occurs in ascitic cirrhotic rats, but its association with ascites infection is unknown. The aim of this study was to assess the relation between bacterial translocation and ascites infection in cirrhotic rats. Male Sprague-Dawley rats were induced to cirrhosis with intragastric CCl4. Ascitic fluid, portal and peripheral blood, mesenteric lymph nodes, liver and spleen samples were cultured before death in those cirrhotic rats with less (group A) or more (group B) than 250 polymorphonuclear neutrophils/mm3 in ascitic fluid, as well as in healthy control rats. Histological examination of jejunum, ileum, and caecum was also performed. Bacterial translocation occurred in 45% of ascitic rats (without differences between groups A and B), but in 0% controls (p = 0.01). Bacterial translocation was associated with positive ascitic fluid culture in 60% of the cases. In all of them the same bacterial species was isolated in both mesenteric lymph node and ascitic fluid. Submucosal caecal oedema (100%), ileal lymphangiectasia (41%), and caecal inflammatory infiltrate (41%) occurred in ascitic rats, the last being associated with ascitic fluid positive culture (p = 0.04). These results suggests that bacterial translocation occurs frequently in ascitic cirrhotic rats, and may play a permissive, but not unique, part in a number of ascites infections. Whether histological changes seen in cirrhotic ascitic rats favour bacterial translocation remains to be elucidated.