In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis

Several clinical trials have revealed that individuals who were given beta-carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150-1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145-1149]. Using differential display techniq…

Identification of a gene-pathway associated with non-alcoholic steatohepatitis.

Background/Aims We have integrated gene expression profiling of liver biopsies of NASH patients with liver samples of a mouse model of steatohepatitis (MAT1A-KO) to identify a gene-pathway associated with NASH. Methods Affymetrix U133 Plus 2.0 microarrays were used to evaluate nine patients with NASH, six patients with steatosis, and six control subjects; Affymetrix MOE430A microarrays were used to evaluate wild-type and MAT1A-KO mice at 15 days, 1, 3, 5 and 8 months after birth. Transcriptional profiles of patients with NASH and MAT1A-KO mice were compared with those of their proficient controls. Results We identified a gene-pathway associated with NASH, that accurately distinguishes betwe…

Nitric oxide triggers mammary gland involution after weaning: remodelling is delayed but not impaired in mice lacking inducible nitric oxide synthase

During mammary gland involution, different signals are required for apoptosis and tissue remodelling. To explore the role of NO in the involution of mammary tissue after lactation, NOS2 (inducible nitric oxide synthase)-KO (knockout) mice were used. No apparent differences were observed between NOS2-KO and WT (wild-type) animals during pregnancy and lactation. However, upon cessation of lactation, a notable delay in involution was observed, compared with WT mice. NOS2-KO mice showed increased phosphorylation of STAT (signal transducer and activator of transcription) 5 during weaning, concomitant with increased beta-casein mRNA levels when compared with weaned WT glands, both hallmarks of th…

El alcance de la responsabilidad solidaria prevista en el artículo 42 ET

El artículo 42 ET impone sobre el empresario que contrata o subcontrata con otros la realización de obras o servicios pertenecientes a su propia actividad una responsabilidad solidaria sobre ciertas obligaciones contraídas por el contratista y el subcontratista con sus trabajadores, siempre que hayan nacido durante la vigencia de la contrata. En principio, el significado de la previsión normativa se presenta como relativamente sencillo; no obstante, su aplicación práctica suscita diferentes dudas interpretativas. Pues bien, el objeto de este trabajo se dirige a perfilar el contorno y contenido de la responsabilidad solidaria recogida en el art. 42 ET, lo que exige delimitar con claridad su …

Hepatocyte growth factor induces MAT2A expression and histone acetylation in rat hepatocytes: role in liver regeneration 1

SPECIFIC AIMSWe have studied the molecular mechanisms and mediators behind the induction of methionine adenosyltransferase 2 A (MAT2A) gene expression in the regenerating rat liver after partial he...

In vivo GSH depletion induces c-myc expression by modulation of chromatin protein complexes.

Abstract We hypothesize that glutathione (GSH) fluctuations could have a prominent role in the modulation of c-myc expression through a mechanism affecting chromatin remodeling complexes. This could lead to an open chromatin structure accessible to transcription factors. We studied the in vivo effect of GSH depletion on these complexes bound to the c-myc promoter in the liver of l-buthionine-(S,R)-sulfoximine (BSO)-treated rats. Using chromatin immunoprecipitation we found that 3 h after BSO treatment the repressing complexes Id2 and Sin3A (part of a histone–deacetylase complex) were released from the c-myc promoter. STAT3 was phosphorylated and associated with its coactivator p300 with int…

Vitamin A deficiency causes oxidative damage to liver mitochondria in rats.

Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three different groups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient rats re-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control and vitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, the mitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7, 8-dihydro-2'-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restored i…

Vitamin E deficiency induces liver nuclear factor-κB DNA-binding activity and changes in related genes

The biological functions of vitamin E have been classically attributed to its property as a potent inhibitor of lipid peroxidation in cellular membranes. However, in 1991, Azzi's group first described that alpha-tocopherol inhibits smooth muscle cell proliferation in a protein kinase C (PKC)-dependent way, demonstrating a non-antioxidant cell signalling function for vitamin E. More recently, the capacity of alpha-tocopherol to modulate gene expression with the implication of different transcription factors, beyond its antioxidant properties, has also been established. This study was to determine the effect of vitamin E-deficiency on liver nuclear factor-kappa B (NF-kappaB) DNA-binding activ…

Novedades jurisprudenciales en materia de transmisión de empresa: la labor del TJUE y del TS en los últimos dos año

Una institución que ha originado una notable atención en los últimos años es la de la transmisión de empresa. En efecto, aún tratándose de un tema “tradicional” en el Derecho del Trabajo, asistimos a una especie de “interés renovado” por el mismo que seguramente derive de los pronunciamientos judiciales dictados en la materia y que han aportado nuevas perspectivas de análisis, tanto desde una óptica general, como desde otra más específica. El objeto de este trabajo se dirige a dar cuenta de las principales “novedades jurisprudenciales” habidas en este terreno

Pandemia y reformas procesales: la incidencia del COVID-19 en el proceso labora

Uno de los efectos de la declaración del estado de alarma por el COVID-19 fue la suspensión de las actividades judiciales. La reanudación de estas actividades implicaba una serie de nuevos retos (acumulación de trabajo; incremento de la litigiosidad; adopción de medidas de seguridad y salud). El RDL 16/2020, de 28 de abril, afrontó algunos de estos retos e introdujo modificaciones tanto en el desarrollo de la actividad judicial, como en la organización de sus recursos. Este trabajo trata de sistematizar tales medidas desde una perspectiva crítica.

Doctrina. La embriaguez y la toxicomanía como causas de despido disciplinario

En el presente trabajo se aborda la embriaguez y la toxicomanía como causas de despi¬do improcedente en el Estatuto de los Trabajadores. A tal efecto se analizan las conduc¬tas básicas que dan pie a tal despido, los requisitos necesarios para que se produzca y la prueba de su incumplimiento. También se realiza una referencia a dichas causas de despido en los Convenios Colectivos.

Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione

In the lactatingmammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21cip1 and p27kip1, cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactatingmammary gland when propargylglycine, an inhibitor of th…

El "genoma" laboral: orígenes, componentes y evolución del derecho del trabajo

El presente trabajo ofrece una reflexión sobre el origen y la evolución del Derecho del Trabajo. El tema, tratado en numerosas ocasiones por la doctrina, se aborda desde una perspectiva distinta a la habitual. Así, siguiendo el ejemplo del profesor Romagnoli, se recurre a un símil consistente en plantear el análisis como un intento por reconstruir lo que podría denominarse �genoma� laboral. Ello lleva a abordar, en primer lugar, el �caldo de cultivo� en el que se gesta, es decir, el contexto histórico en el que aparece esta disciplina; en segundo lugar, sus �cromosomas�, es decir, los distintos componentes que la integran y modelan; finalmente, los �agentes mutágenos�, es decir, las circuns…

Differential functions of calpain 1 during epithelial cell death and adipocyte differentiation in mammary gland involution

Calpains become activated in the mammary gland early during weaning, cleaving several proteins located mainly in the cell membrane, but also in other organelles such as lysosomes, mitochondria and nuclei. By immunofluorescence and Western blot analysis, we have demonstrated the nuclear translocation of calpain-1 and calpain-2, together with the cleavage of several cytoplasmic nucleoporins in epithelial cells of the lobulo-alveolar compartment. In vivo and in vitro calpain inhibition prevented this nucleoporin degradation. In addition, calpain-1 was also present in the nucleus of non-epithelial mammary tissue cells, concomitant with adipocyte re-differentiation. Calpain-1 was internalized wi…

Fatty Liver and Fibrosis in Glycine N-Methyltransferase Knockout Mice Is Prevented by Nicotinamide

Deletion of glycine N-methyltransferase (GNMT), the main gene involved in liver S-adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5-month-old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, …

Nitration of cathepsin D enhances its proteolytic activity during mammary gland remodelling after lactation

Proteomic studies in the mammary gland of control lactating and weaned rats have shown that there is an increased pattern of nitrated proteins during weaning when compared with controls. Here we report the novel finding that cathepsin D is nitrated during weaning. The expression and protein levels of this enzyme are increased after 8 h of litter removal and this up-regulation declines 5 days after weaning. However, there is a marked delay in cathepsin D activity since it does not increase until 2 days post-weaning and remains high thereafter. In order to find out whether nitration of cathepsin D regulates its activity, iNOS (inducible nitric oxide synthase)−/− mice were used. The expression…

Retinoids induce MMP-9 expression through RARalpha during mammary gland remodeling.

Retinoic acid (RA) is a signaling molecule in the morphogenesis of the mammary gland, modulating the expression of matrix metalloproteinases (MMPs). The aim of this paper was to study the role of RA during weaning, which consists of three events: apoptosis of the secretory cells, degradation of the extracellular matrix, and adipogenesis. CRABP II and CRBP-1 carrier proteins increased significantly during weaning compared with lactating glands but reverted to control values after the litter resuckled. The effects of RA are mediated by the nuclear receptors RARalpha, RARbeta, RARgamma, and RXRalpha, which underwent an increase in protein levels during weaning. In an attempt to elucidate the R…

NF-ĸB as node for signal amplification during weaning.

Post-lactational involution has been reported to share common features with breast tumor development. A deep characterization of the signaling triggered after weaning would help to unveil the complex relationship between involution and breast cancer. NF-κB, a crucial factor in the involuting gland, might be an important regulatory node for signal amplification after weaning; however there is limited information about the identity of NF-κB-target genes and the molecular mechanisms leading to the selection of genes involved in a particular biological process. We identified 4532 target genes in mammary gland at 48h weaning, by genome-wide analysis of regions bound by RelA(p65)-NF-κB in vivo. I…

La simulación de conflictos en la enseñanza del Derecho Procesal Laboral

Weaning induces NOS-2 expression through NF-κB modulation in the lactating mammary gland: importance of GSH

Zaragozá, R; Miralles, VJ; Rus, AD; García, C; Carmena, R; García-Trevijano, ER; Barber, T; Pallardó, FV; Torres, L; Viña, JR. At the end of lactation the mammary gland undergoes involution, a process characterized by apoptosis of secretory cells and tissue remodelling. To gain insight into this process, we analysed the gene expression profile by oligonucleotide microarrays during lactation and after forced weaning. Up-regulation of inflammatory mediators and acute-phase response genes during weaning was found. Expression of IκBα (inhibitory κBα), a protein known to modulate NF-κB (nuclear factor-κB) nuclear translocation, was significantly up-regulated. On the other hand, there was a time-…

Molecular mechanisms of Id2 down-regulation in rat liver after acetaminophen overdose. Protection by N-acetyl-L-cysteine.

Id2 is a pleiotropic protein whose function depends on its expression levels. Id2-deficient cells show increased cell death. This study explored the molecular mechanisms for the modulation of Id2 expression elicited by GSH and oxidative stress in the liver of acetaminophen (APAP)-intoxicated rats. APAP-overdose induced GSH depletion, Id2 promoter hypoacetylation, RNApol-II released and, therefore, Id2 down-regulation. Id2 expression depends on c-Myc binding to its promoter. APAP-overdose decreased c-Myc content and binding to Id2 promoter. Reduction of c-Myc was not accompanied by decreased c-myc mRNA, suggesting a mechanism dependent on protein stability. Administration of N-acetyl-cystein…

Glutamate cysteine ligase up-regulation fails in necrotizing pancreatitis

Glutathione depletion is a key factor in the development of acute pancreatitis. Our aim was to study the regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in edematous or necrotizing pancreatitis in rats. Glutathione levels were kept low in necrotizing pancreatitis for several hours, with no increase in protein or mRNA levels of glutamate cysteine ligase subunits, despite binding of RNA polymerase II to their promoters and coding regions. The survival signal pathway mediated by ERK and c-MYC was activated, and c-MYC was recruited to the promoters. The failure in gene up-regulation seems to be due to a marked increase in cytosolic ribonuclease activi…

Id2 leaves the chromatin of the E2F4-p130-controlled c-myc promoter during hepatocyte priming for liver regeneration

The Id (inhibitor of DNA binding or inhibitor of differentiation) helix–loop–helix proteins are involved in the regulation of cell growth, differentiation and cancer. The fact that the molecular mechanisms of liver regeneration are not completely understood prompted us to study the fate of Id2 in proliferating liver. Id2 increases in liver regeneration after partial hepatectomy, following the early induction of its gene. Co-immunoprecipitation shows that Id2 forms a complex with E2F4, p130 and mSin3A in quiescent liver and all these components are present at the c-myc promoter as shown using ChIP (chromatin immunoprecipitation). Activation of c-myc during hepatocyte priming (G0–G1 transitio…

RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.

We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to thos…

Blockade of the trans-sulfuration pathway in acute pancreatitis due to nitration of cystathionine β-synthase

© 2019 Published by Elsevier B.V.

Vitamin A Deficiency and the Lung.

Vitamin A (all-trans-retinol) is a fat-soluble micronutrient which together with its natural derivatives and synthetic analogues constitutes the group of retinoids. They are involved in a wide range of physiological processes such as embryonic development, vision, immunity and cellular differentiation and proliferation. Retinoic acid (RA) is the main active form of vitamin A and multiple genes respond to RA signalling through transcriptional and non-transcriptional mechanisms. Vitamin A deficiency (VAD) is a remarkable public health problem. An adequate vitamin A intake is required in early lung development, alveolar formation, tissue maintenance and regeneration. In fact, chronic VAD has b…

La extinción del contrato de trabajo en la reforma laboral de 2012

Una de las instituciones claves en todo sistema de relaciones laborales es, sin lugar a dudas, la extinción del contrato de trabajo. Ese carácter fundamental seguramente explique que su regulación sea objeto de una continua atención en diversos planos, no solo en el normativo, sino también en el político y en el doctrinal. La reforma laboral de 2012 ha afectado de manera notable a la materia extintiva. Pues bien, el objeto de este trabajo se dirige a dar cuenta de algunos de estos cambios: de entrada, se analizan los producidos en el plano causal y procedimental, lo que remite al análisis de la reforma en los arts. 51 y 52 ET; a continuación se abordan los cambios en el régimen de efectos, …

Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma

Abstract Background/Aims: It has been known for at least 50 years that alterations in methionine metabolism occur in human liver cirrhosis. However, the molecular basis of this alteration is not completely understood. In order to gain more insight into the mechanisms behind this condition, mRNA levels of methionine adenosyltransferase ( MAT1A ), glycine methyltransferase ( GNMT ), methionine synthase ( MS ), betaine homocysteine methyltransferase ( BHMT ) and cystathionine β-synthase ( CBS ) were examined in 26 cirrhotic livers, five hepatocellular carcinoma (HCC) tissues and ten control livers. Methods: The expression of the above-mentioned genes was determined by quantitative RT-PCR analy…

Role of GSH in the modulation of NOS-2 expression in the weaned mammary gland

GSH delivery to the lactating mammary gland is essential for the maintenance of lactation as its decrease leads to apoptosis and involution of the mammary gland. In fact, it has already been demonstrated that some of the changes in gene expression found in the lactating mammary gland after forced weaning are reproduced in rats treated with buthionine sulphoximine to deplete GSH levels. An oligonucleotide microarray experiment would give us a better knowledge of the mRNA expression patterns during lactation and after weaning and the possible functions of GSH in the modulation of these events.

Chromatin structure of the Arabidopsis thaliana Adh promoter: analysis of nucleosome positioning

Abstract The chromatin structure of the promoter and proximal 5′ transcribed region of the Arabidopsis thaliana Adh gene has been studied in three experimental models: whole plants under aerobic conditions in which the gene is repressed, whole plants under flooding-induced anaerobiosis, in which the expression of the gene occurs in some plant tissues and yeast cells in which the Arabidopsis Adh DNA had been cloned but is not expressed at all. Experiments of indirect end labelling after DNase I and micrococcal nuclease digestion of nuclei allowed us to conclude that no positioned nucleosomes exist in plant nuclei on the proximal region of the promoter (up to −350), probably due to the presen…

Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.

Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved produc…

Elevated Expression of Liver γ-Cystathionase Is Required for the Maintenance of Lactation in Rats

Liver gamma-cystathionase activity increases in rats during lactation; its inhibition due to propargylglycine is followed by a significant decrease in lactation. This is reversible by N-acetylcysteine administration. To study the role of liver gamma-cystathionase and the intertissue flux of glutathione during lactation, we used lactating and virgin rats fed liquid diets. Virgin rats were divided into two groups as follows: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet-fed rats); the other virgin group was fed the normal liquid diet (control). The expression and activity of liver gamma-cystathionas…

Transcription of the MAT2A gene, coding for methionine adenosyltransferase, is up-regulated by E2F and Sp1 at a chromatin level during proliferation of liver cells

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes. Here, we report studies on the molecular mechanisms controlling the induction of MAT2A in regenerating rat liver and in proliferating hepatocytes. The MAT2A is up-regulated at two discrete moments during liver regeneration, as confirmed by RNApol-ChIP analysis. The first one coincides with hepatocyte priming (i.e. G0-G1 transition), while the second one tak…

Isolation and Characterization of an Fe(III)-Chelating Compound Produced by Pseudomonas syringae

The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, δ- N -hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10 32 ) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by an…