The yield strength of different pipeline steel grades, rolled under four different conditions, was correlated with calculated strengthening contributions. Slabs with the same composition were rolled ...under identical roughing conditions but varied finish rolling temperature (FRT). Two cooling routes, consisting of accelerated water cooling condition (ACC) followed by slow cooling in an oven to simulate coiling and air cooling were applied after the last rolling pass. The microstructures obtained after each thermo mechanical controlled process (TMCP) schedule, were characterized using Transmission Electron Microscopy (TEM), Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Electron backscatter diffraction (EBSD). The mechanical properties of the plates were determined by means of tensile tests and Charpy V-notch impact test. It was confirmed that a combination of fast cooling rate and low finish rolling temperature produces higher strength than the slow cooling rate and high finish rolling temperature. Contributions to the strengthening arising from the various microstructural features like solid solution strengthening, grain size, dislocation density and precipitation hardening, were analyzed using Taylor, Hall–Petch and Ashby–Orowan approaches. The root of the sum of the squares method was applied to link the experimental with the model-predicted strength. It is believed that this approach provides a better understanding of the effect of TMCP parameters on the microstructure and strengthening mechanisms in pipeline steels.
This paper reports the friction and wear response of WC–10%Co(Cr/V) cemented carbide with different surface finishes, attained by grinding (G) and wire-EDM, respectively, during sliding experiments ...at 400°C. For comparison, tests under the same conditions were carried out at 25°C. The wear experiments were performed under a normal force of 14N, which produced a Hertzian maximum pressure of 3.10GPa, and a sliding speed of 0.3m/s against WC–6%Co(Cr/V) balls of 6mm diameter. At 25°C the average values of the friction coefficients were 0.36±0.04 and 0.39±0.06 for the ground and wire-EDM surface finishes, respectively. The mechanical behavior of both systems at 25°C was assessed by carrying out analytical calculations of the stress field created by a circular sliding contact under a spherical indenter, where the residual stresses were considered. The theoretical results are in agreement with the experimental data, indicating that the wire-EDM sample has a specific wear rate, which is approximately 3.1 times greater than that corresponding to the G sample at 25°C. At 400°C, an increase in the friction coefficients takes place up to values of 0.75±0.1 and 0.71±0.8, for the ground and wire-EDM surface finishes, respectively. The increase was associated to an adhesive mechanism, which is more pronounced for the G sample. However, for the wire-EDM sample this increase was more linked to a marked abrasive mechanism. The wear rates for both samples at 400°C are similar to those obtained at 25°C, which indicates that apparently the test temperature does not have an important effect on the wear rate. However, it is known that temperature influences considerably the residual stress nature. Therefore, these results were explained by taking into account the wear mechanisms between the tribopairs in view of the mechanical characteristics and the morphological features obtained from SEM coupled with EDS analysis.
•The effect of machining on the friction of cemented carbide tribopairs has been investigated.•A correlation between surface integrity, sliding contact and residual stresses has been established.•The wear mechanism of cemented carbides with different properties has been studied at 25 and 400°C.•The morphological features of the wear tracks obtained by means of SEM-EDS, have been analyzed.•The relationship between the critical flaw size of the grinded and wire-EDM samples has been established.
Background & Aims: Oxygen supply to the hepatic parenchyma is compromised by long- or short-term ethanol consumption and pathological conditions such as cirrhosis. Impairment in the production of
...S-adenosyl-
L-methionine, the major methylating agent, occurs during hypoxia. In this study, the molecular mechanisms implicated in the regulation of
S-adenosyl-
L-methionine synthesis by oxygen levels were investigated.
Methods: Rat hepatocytes were isolated and cultured under normoxic (21% O
2) or hypoxic (3% O
2) conditions for different periods. Methionine adenosyltransferase activity, messenger RNA levels, and nuclear transcription were evaluated.
Results: Methionine adenosyltransferase was inactivated in hepatocytes kept under low oxygen levels. Hypoxia induced the expression of nitric oxide (NO) synthase, and the inactivation of methionine adenosyltransferase was prevented by the NO synthase inhibitor
N
G
-monomethyl-
L-arginine methyl ester. Methionine adenosyltransferase messenger RNA levels were down-regulated by hypoxia, through a mechanism that might involve a hemoprotein. Hypoxia dramatically reduced methionine adenosyltransferase gene transcription, and messenger stability was also decreased, although to a lesser extent.
Conclusions: We have established the molecular basis for the regulation of methionine adenosyltransferase activity and gene expression by hypoxia. NO-mediated inactivation and transcriptional arrest seem to be the two major pathways by which oxygen levels control hepatic methionine adenosyltransferase, the enzyme necessary for methylation reactions and for the synthesis of polyamines and glutathione.
GASTROENTEROLOGY 1998;114:364-371
ABSTRACT
Methionine metabolism starts with the formation of S‐adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase ...(MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver‐specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose‐dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3‐deazaadenosine and L‐ethionine, but not D‐ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte.—García‐Trevijano, E. R., Ujue Latasa, M., Victoria Carretero, M., Bera‐sain, C., Mato, J. M., and Avila, M. A. S‐Adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S‐adenosylme‐thionine in the maintenance of the differentiated status of the liver. The FASEB J. 14, 2511–2518 (2000)
Basiliximab (Simulect) is a high-affinity chimeric and humanized monoclonal antibody, directed against the alpha chain of human interleukin-2 receptor (CD25). The administration of two doses (20 mg ...intravenously per dose), one given 2 hours before transplantation and the second on day 4 posttransplant, provides suppression of the interleukin-2 receptor for up to 45 days, reducing the rate of acute rejection in kidney transplantation. This study was designed to compare the efficacy of a single dose of Simulect to the recommended two doses. The other objective was the reduction of the costs related to the standard two dose protocol. Fifty-two patients were included: group I (32 patients) received two doses of Simulect; group II (20 patients) received one dose. There were 39 living related donors and 13 living unrelated. All patients were followed for 1 year. Maintenance immunosuppression consisted of tacrolimus or cyclosporine, mycophenolate mofetil, and steroids. The diagnosis of rejection was made clinically. All episodes were treated with intravenous steroids. The incidence of rejection was similar in both groups; there was no graft loss to rejection. There were two deaths in group I, and one death in group II, yielding graft and patient actual survival rates at 1 year of 93% and 95%, respectively. These results suggest that Simulect is equally effective when administered in two doses or in a single dose in kidney transplantation. The reduction of cost by giving a single dose is significant, especially in developing countries without national health insurance.
Although free radicals have been traditionally implicated in cell injury, and associated to pathophysiological processes, recent data implicate them in cell signaling events. Free radicals are ...naturally occurring oxygen‐, nitrogen‐ and sulfur‐derived species with an unpaired electron, such as superoxide, hydroxyl radical or nitric oxide. In order to assess the role of free radicals in cell signaling, we have studied the modulator effect of oxygen and nitrogen active species on liver methionine adenosyltransferase (MAT), a key metabolic enzyme. The presence of 10 cysteine residues per subunit, makes liver MAT a sensitive target for oxidation/nitrosylation. Here we show that purified MAT from rat liver is nitrosylated and oxidized in vitro. Incubation with H2O2 or the NO donor S‐nitrosylated GSH (GSNO), diminish MAT activity in a dose‐ and time‐dependent manner. Furthermore, the inactivation derived from both oxidation and nitrosylation, was reverted by GSH. MAT inactivation originates on the specific and covalent modification of the sulphydryl group of cysteine residue 121. We also studied how free radicals modulate MAT activity in vivo. It was previously shown that MAT activity is strongly dependent on cellular GSH levels. Generation of oxygen and nitrogen active species in rats by injection of LPS, induced a decrease of liver MAT activity. This effect might derive from nitrosylation and/or oxidation of the enzyme. Modulation of liver MAT by NO is further supported by the inactivation of this enzyme observed in experimental models in which NO is produced; such as the administration of NO donors to rats and in hepatocytes cultured in hypoxia, a condition that induces the expression of the inducible nitric oxide synthase (iNOS). Oxidation also controls liver MAT activity in a cell environment as shown in CHO cells stably transfected with rat liver MAT cDNA upon addition of H2O2 to the culture medium. This effect depends upon the generation of the hydroxyl radical. On the basis of the metabolic implications of liver MAT, together with the structural features accounting for the sensitivity of this enzyme to active oxygen and nitrogen species, we propose that modulation of MAT by these agents could be a mechanism to regulate the consumption of ATP in the liver, and thus preserve cellular viability under different stress conditions.
ABSTRACT
Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S‐adeno‐sylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product ...of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A. The mechanisms behind the tissue and differentiation state specific MAT1A expression are not known. In the present work we examined MAT1A promoter methylation status by means of methylation sensitive restriction enzyme analysis. Our data indicate that MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and fetal rat hepatocytes, indicating that this modification is tissue specific and developmentally regulated. Immunoprecipitation of mononucleosomes from liver and kidney tissues with antibodies mainly specific to acetylated histone H4 and subsequent Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chromatin acetylation. Early changes in MAT1A methylation are already observed in the precancerous cirrhotic livers from rats, which show reduced MAT1A expression. Human hepatoma cell lines in which MAT1A is not expressed were also hypermethylated at this locus. Finally we demonstrate that MAT1A expression is reactivated in the human hepatoma cell line HepG2 treated with 5‐aza‐2’‐deoxycytidine or the histone deacetylase inhibitor trichosta‐tin, suggesting a role for DNA hypermethylation and histone deacetylation in MAT1A silencing.—Torres, L., Åvila, M. A., Carretero, M. V., Latasa, M. U., Caballería, J., López‐Rodas, G., Boukaba, A., Lu, S. C., Franco, L., Mato, J. M. Liver‐specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation. FASEB J. 14, 95–102(2000)
Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular ...mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated α1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.
Uncoupling protein 2 (UCP2) expression in liver is restricted to non-parenchymal cells. By means of differential display screening between normal rat liver and H4IIE hepatoma cells we have isolated a ...cDNA clone encompassing part of UCP2 cDNA. Northern blot analysis revealed that UCP2 is expressed in some hepatocarcinoma cell lines, while it is absent in adult hepatocytes. UCP2 mRNA in H4IIE cells was downregulated when cells were cultured for 36 h in 0.1% serum and its expression was restored upon addition of 10% serum or phorbol esters. Hypomethylation of
UCP2 was observed in transformed UCP2 expressing cells. Our results indicate that UCP2 is expressed in some hepatocarcinoma cell lines and that serum components may participate in maintaining elevated UCP2 levels.
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