Biomedical subjects
Publications and source records attributed to M Benito.
The effect of TNF-alpha on cell death in rat fetal brown adipocytes maintained in primary culture was determined. TNF-alpha inhibited proliferation and induced apoptosis in these cells. Most of the cells undergoing apoptosis after TNF-alpha treatment did not express PCNA, suggesting an induction of apoptosis by TNF-alpha in non-proliferative cells. IGF-I but not EGF prevented TNF-alpha-induced apoptosis.
We have previously reported that transforming growth factor-beta (TGF-beta) induces apoptosis in fetal hepatocytes in primary culture. This effect was found to be associated with an increase in intracellular reactive oxygen species (ROS) and a lowering of total cellular reduced glutathione (GSH). In this study, we investigated whether protein synthesis plays a role in these TGF-beta-induced effects. When fetal hepatocytes were incubated in the presence of cycloheximide, a specific protein synthesis inhibitor, TGF-beta-induced apoptosis was completely blocked. The overall intracellular oxidized state of the cells, when measured using either 2',7'-dichlorofluorescein diacetate (DCFH) by laser-scanning confocal microscopy, or both DCFH and hydroethidine (DHE) by flow cytometric analysis, was increased transiently after the addition of TGF-beta. This increase was abolished by incubation of the cells in the presence of cycloheximide. Furthermore, the decrease in the total cellular GSH content induced by TGF-beta in these cells was not observed when cycloheximide was present. Cycloheximide effect was not associated with an enhancement of cysteine and restoration of cellular glutathione level, because inhibition of GSH synthesis with buthionine sulfoximine (BSO) did not prevent the cycloheximide protective effect. Experiments performed to check whether Fas (APO-1) ligand might be the protein that needs to be synthesized have indicated that this possibility can be excluded. All these findings suggest that apoptosis elicited by TGF-beta in fetal hepatocytes requires the synthesis of an unknown protein before ROS production, glutathione loss, and oxidative stress.
Inactivation of the tumour-suppressor gene p53 has been described as one of the most common molecular changes found in lung tumours. Our purpose was to study the prognostic value of p53 alterations and to determine whether some specific mutation type in the p53 gene could be associated with poor clinical evolution in non-small-cell lung cancer (NSCLC) patients. To this end, we studied 81 resected primary NSCLCs in order to detect p53 alterations. p53 protein accumulation was analysed using immunohistochemistry methods; p53 gene mutations in exons 5-9 were studied using polymerase chain reaction-single-strand conformation polymorphism and sequencing techniques. p53 protein was immunodetected in 46.9% of lung carcinomas and 44.7% of p53-immunopositive tumours showed p53 mutations. Survival analysis was performed on 62 patients. No survival differences were found for patients with or without p53 immunopositivity. A shorter survival was found in patients with underlying p53 gene mutations, mainly in patients with squamous cell lung tumours; the worst prognosis was found when mutations were located in exon 5 (P = 0.007). In conclusion, the location of p53 mutations might be considered as a prognostic indicator for the evaluation of poor clinical evolution in NSCLC patients.
The cirrhogenic ability of thioacetamide has been used to induce a model of chronic generalized liver disease that resembles the preneoplastic state of human fibrosis. Malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH) are two cytosolic NADPH-generating enzymes; their activities significantly increased in liver when macronodular cirrhosis was induced by long-term thioacetamide administration to rats. The progressive increase in G6PDH and ME activities during the cirrhogenic process is parallel to the induction in gene expression of both enzymes detected by the increase in their mRNAs. These data indicate that NADPH-consuming mechanisms such as the microsomal oxidizing system and the maintenance of the cell redox state could be involved. A relationship between the extent of G6PD and ME gene expression and oxidative stress generated by the oxidative metabolism of thioacetamide is proposed as the hepatic concentration of malondialdehyde, a metabolite derived from lipid peroxidation, underwent a progressive and significant enhancement during thioacetamide-induced cirrhogenesis. These results led us to suggest that the enhanced activities of G6PDH and ME might be related to microsomal mechanisms of detoxification as well as to the maintenance of the cellular redox state. Furthermore, the noticeable increase in the hepatocyte population involved in DNA replication parallel to G6PDH activity suggests that G6PDH, through ribose-5-phosphate, might also be involved in the processes of DNA synthesis and repair.
In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.
In the present study we have examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the insulin-like growth factor I (IGF-I)-signaling pathways involved in differentiation and in mitogenesis in fetal rat brown adipocytes. Activation of PI 3-kinase in response to IGF-I was markedly inhibited by two PI 3-kinase inhibitors (wortmannin and LY294002) in a dose-dependent manner. IGF-I-stimulated glucose uptake was also inhibited by both compounds. The expression of adipogenic-related genes such as fatty acid synthase, malic enzyme, glycerol 3-phosphate dehydrogenase, and acetylcoenzyme A carboxylase induced by IGF-I was totally prevented in the presence of IGF-I and any of those inhibitors, resulting in a marked decrease of the cytoplasmic lipid content. Moreover, the expression of the thermogenic marker uncoupling protein induced by IGF-I was also down-regulated in the presence of wortmannin/LY294002. IGF-I-induced adipogenic- and thermogenic-related gene expression was only partly inhibited by the p70S6k inhibitor rapamycin. In addition, pretreatment of brown adipocytes with either wortmannin or LY294002, but not with rapamycin, blocked protein kinase C zeta activation by IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation was PI 3-kinase-independent. Our results show for the first time an essential requirement of PI 3-kinase in the IGF-I-signaling pathways leading to fetal brown adipocyte differentiation, but not leading to mitogenesis. In addition, protein kinase C zeta seems to be a signaling molecule also involved in the IGF-I differentiation pathways downstream from PI 3-kinase.
Fetal rat brown adipocytes show high-affinity binding sites for both insulin-like growth factor I (IGF-I) and insulin. Cell culture for 24 h in the presence of IGF-I or insulin, independently, up-regulated the mRNA expression of adipogenic-related genes, such as fatty acid synthase (FAS), glycerol-3-phosphate de-hydrogenase and insulin-regulated glucose transporter Glut4, and down-regulated the expression of phosphoenolpyruvate carboxykinase mRNA in a dose-dependent manner. Moreover, both IGF-I and insulin increased the FAS gene transcription rate at 2 h, producing a time-dependent accumulation of FAS mRNA. Furthermore IGF-I or insulin increased glucose uptake and lipid content throughout the 24 h culture period. Our results suggest that both IGF-I and insulin are major signals involved in initiating and/or maintaining the expression of adipogenic-related genes in fetal rat brown adipocytes.
We studied whether the TGF-beta-induced apoptosis in fetal hepatocyte primary cultures may be modulated by the presence of mitogenic stimuli, such as EGF or HGF. EGF prevented cell death, showing a dose dependence that was identical to that observed for its effect on DNA synthesis stimulation. HGF, in contrast, had no effect, even at high concentrations. EGF blocked apoptosis, since in the presence of this factor cells did not show DNA fragmentation. Moreover, EGF, but not HGF, blocked c-fos induction associated with the apoptotic process induced by TGF-beta in these cells.
Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose-response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos-induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes.
Confluent fetal rat brown adipocytes in primary culture showed an almost undetectable level of uncoupling protein (UCP) mRNA and a low mitochondrial content of functional UCP. Treatment of confluent cells with 10 nM triiodothyronine in a serum-free medium, in the absence of noradrenergic stimulation, increased the amount of UCP mRNA in a time-dependent manner. This effect was due to an increased UCP gene transcription rate and UCP mRNA stabilization, resulting in a higher content of immunoreactive mitochondrial UCP and functional UCP (detected by its ability to bind GDP). Thus, triiodothyronine might play a significant physiological role in the UCP expression throughout fetal development, when brown adipose tissue starts to differentiate and UCP is primarily expressed.
The presence of transforming growth factor beta 1 (TGF-beta 1) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G2/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-beta 1 (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-beta 1 for 48 h increased by 5-fold the percentage of cells containing (3H)thymidine-labeled nuclei as compared to quiescent cels. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (3H)thymidine-labeled nuclei in response to TGF-beta 1. Moreover, TGF-beta 1 induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and beta-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-beta 1 for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-beta 1 induces the expression of very early genes related to cell proliferation, TGF-beta 1 could be acting either as a mitogen or as a survival factor in induce proliferation to fetal brown adipocytes.
Chronic liver damage induced by thioacetamide (TAM) was accompanied by changes in the expression of genes related to growth (beta-actin) and function (albumin and haptoglobin) of the liver. Their messenger RNA (mRNA) levels increased during the first days after TAM administration, but 4 to 7 days after prolonged treatment with this drug, liver gene expression was considerable decreased. TAM-induced changes in albumin and beta-actin mRNA levels were prevented by cotreatment with S-adenosyl-L-methionine (SAM). We have investigated the possible involvement of glutathione in the protective mechanism of SAM. Firstly, we found that TAM treatment in the rat induced changes in liver glutathione disulfide (GSSG) levels, with a concomitant increase in the glutathione reductase enzymatic activity, these changes being abolished when animals were cotreated with TAM and SAM. Secondly, when rats were pretreated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, before thioacetamide administration, the beneficial effect of SAM on liver gene expression was completely abolished. These results were confirmed by assaying the alanine transaminase serum activity, a parameter of liver injury. TAM-treated animals had increases in this serum enzyme, this effect being partially blocked by SAM. However, in BSO-pretreated rats, the protective effect of SAM was impaired. Taking together all these results, we propose a glutathione-dependent mechanism in the SAM protection against TAM hepatotoxicity in the rat.
Between April 1987 and October 1992, six cases of Brucella endocarditis were operated on in the authors' hospital for valve replacement. They were five men and one woman with a mean(s.d.) age of 52(15) years (range 30-71 years). Three patients were in New York Heart Association (NYHA) class III and three in class IV. Two patients had previous history of rheumatic fever, one was a drug abuser, two had peripheral embolism and one constrictive pericarditis. Most were living in rural areas. Echocardiographic diagnoses were: severe aortic regurgitation in two patients, mixed disease in two and double valve involvement in two. Valve vegetations were demonstrated in two patients, valve calcification in two and annulus abscess in two others. Serological tests were positive in all patients. All patients had valve replacements and three were operated on as emergencies. Surgical findings were: valve vegetations in two patients, cusp perforation in two annulus abscess in two and prosthetic leak in two. Mean(s.d.) cardiopulmonary bypass time was 123(77) min with a mean ischaemic time of 79(43) min. All patients were given specific antibiotic treatment after surgery. There was no intraoperative mortality and the 5-year survival rate was 100%. Early reoperations were needed in three patients, two because of prosthetic leakage causing severe regurgitation and one for tamponade. The results suggests that Brucella endocarditis is rare, but still occurs in Mediterranean areas. Surgical replacement is needed in spite of antibiotic treatment and recurrences with prosthetic leaks are usual.
The main source of insulin-like growth factor I (IGF-I) postnatally is the liver, under growth hormone stimulation, although IGF-I is already present in embryonic tissues and in fetal serum, when its expression is independent of growth hormone. The extracellular alpha-subunit of the IGF-I receptor (IGF-IR) contains an IGF-I binding domain, and the beta-subunit possesses tyrosine kinase activity, which is greatly enhanced when IGF-I binds to the alpha-subunit and leads to its autophosphorylation. Insulin receptor substrate 1 (IRS-1) is the most well characterized cellular substrate for IGF-I, containing at least 20 potential tyrosine phosphorylation sites. The tyrosine phosphorylated form of IRS-1 acts as a docking protein by associating SH2-containing proteins including the p85 regulatory subunit of phosphatidylinositol-3-kinase (P13-kinase), the protein tyrosine phosphatase SH-PTP2, the SH2- and SH3-containing adaptor protein Nck and the growth factor receptor-bound protein-2 (Grb2/Sem5) protein. Grb2 is found associated with mSOS, a GTP/GDP exchange factor involved in converting the inactive Ras-GDP to the active Ras-GTP. The p85 regulatory subunit of PI3-kinase can be also a direct in vitro substrate of the IGF-IR. Although IRS-1 is the major substrate of the IGF-IR, there is another early phosphotyrosine substrate termed SHC, which also activates Ras via Grb2-mSos complex. Activation of p21-Ras induces a serine/threonine kinase cascade leading to the activation of MAP-kinases. The importance of IGF-I as a mitogen throughout development has been clearly demonstrated in IGF-I and IGF-IR knockout mouse studies and also in transgenic mice over-expressing IGF-I. IGF-I is a mitogen in many cell types in culture such as T lymphocytes, chondrocytes or osteoblasts and it is considered to be a progression factor in mouse fibroblasts. IGF-I is also involved in muscle, neurons and adipogenic differentiation of mesenchymal cells. However, IGF-I induces proliferation and differentiation in fetal brown adipocytes, suggesting that both cellular processes are not necessarily mutually exclusive in fetal cells.
BACKGROUND/AIMS: Hepatic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) gene is absent in fetal liver. However, this gene can be initially expressed in 20-day-old fetal hepatocyte primary cultures under specific hormonal stimulation. The role of transcriptional factors involved is also studied. METHODS: Primary 20-day-old fetal hepatocytes have been cultured and Northern-blot and nuclear run-on transcription assays have been performed. RESULTS: Fetal hepatocytes in culture initially expressed PEPCK gene by dibutyryl cAMP, in the presence of dexamethasone. Dibutyryl cAMP increased by 8-fold the rate of transcription of PEPCK gene at 30 min, and produced a 50-fold increase in its mRNA content at 3 h. This induction of PEPCK expression by cAMP occurred in the presence of sustained levels of CCAAT/enhancer binding protein (C/EBP) alpha-delta mRNAs, and was accompanied by an increase in the rate of transcription and mRNA content of C/EBP beta gene, and a decrease in the expression of c-myc, in the absence of c-fos expression. In addition, insulin or phorbol esters decreased by 50% the PEPCK rate of transcription and its mRNA accumulation induced by dibutyryl cAMP. This inhibitory effect of insulin or phorbol esters on PEPCK gene expression was accompanied by an increase in the rate of transcription and mRNA content of nuclear factors such as c-fos and c-myc, the expression of C/EBPs remaining essentially unmodified.
BACKGROUND: There are no cases described in the medical literature of systemic allergic reactions due to oregano (Origanum vulgare) or thyme (Thymus vulgaris). These herbs belong to the Lamiaceae (Labiatae) family which comprises other plants such as hyssop (Hyssopus officinalis), basil (Ocimum basilicum), marjoram (Origanum majorana), mint (Mentha piperita), sage (Salvia officinalis) and lavender (Lavandula officinalis). OBJECTIVE: We describe three systemic allergic reactions caused by oregano and thyme in the same patient. METHODS: Skin tests with inhalant allergens and plants of the Labiatae family were done. We used the prick by prick technique with dried commercial plants and prick tests with extracts prepared with the Frugoni method in our patient and in ten control patients. Total serum IgE was determined by Phadezym IgE PRIST (Pharmacia). Specific IgE was measured by two methods: CAP system (Pharmacia) and Phadezym RAST (Pharmacia Diagnostics, Uppsala, Sweden) with activated discs of the allergenic extracts that were prepared in our laboratory. RESULTS: Skin tests with inhalants were positive to grasses. Skin tests with plants of the Labiatae family were positive in all cases when the skin prick technique was used; tests were negative with basil and lavender, and positive with all the others when we used the prick by prick technique. We did not detect any positive skin tests nor specific IgE to plants of the Labiatae family in control patients. Total serum IgE was 406 U/mL. Specific IgE was detected to all herbs tested; higher levels were obtained with the CAP system. CONCLUSIONS: Plants belonging to the Labiatae family seem to show cross-sensitivity on the basis of clinical history and in vitro and in vivo test results.
Since Ras proteins are essential intermediates of some insulin-like growth factor I (IGF-I)/insulin signaling pathways, we examined whether Ras proteins mediate the IGF-I-induced uncoupling protein expression. Additionally, the role of Ras proteins on IGF-I and IGF-I receptor expression was studied. IGF-I treatment of fetal brown adipocytes cotransfected with inducible gene constructs of SV40 large T antigen (SV40LTag) and a transforming ras gene induced uncoupling protein expression (UCP) in the absence of expression of the transfected genes. The expression of the dexamethasone-inducible transforming ras gene alone or in combination with the Zn-inducible SV40LTag mimicked the IGF-I effect inducing UCP expression and IGF-I did not induce it further. However, the expression of the Zn-inducible SV40LTag did not increase UCP expression in the absence of IGF-I. Expression of the transfected ras oncogene also induced IGF-I and IGF-I receptor mRNAs, whereas expression of SV40LTag did not increase them. Specific IGF-I binding was also specifically increased by expression of the transfected ras oncogene but was not affected by expression of the SV40LTag construct. These results indicate that Ras proteins mediate the IGF-I-induced effect on UCP expression and play a role in the expression of IGF-I and IGF-I receptor. Therefore, an IGF-I autocrine/paracrine loop might be implicated in the process of thermogenic differentiation of brown adipose tissue by a new mechanism unlike that induced by norepinephrine.
In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.