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Biomedical subjects

M Benito

Publications and source records attributed to M Benito.

At least 91 records · Page 5Linked to original sources
PubMed1996

p21ras induced differentiation-related gene expression in fetal brown adipocyte primary cells and cell lines.

Transfection of primary rat fetal brown adipocytes with constructs of SV40 large T antigen, alone and together with lys12-mutated H-ras gene, gave permanent cell lines showing an immortalized or transformed phenotype, respectively, all of them selected by the expression of the uncoupling protein (UCP), a tissue-specific marker. Primary brown adipocytes and immortalized cell lines respond to insulin-like growth factor I (IGF-I) by increasing their lipid content and the mRNA expression of both the adipogenic marker fatty acid synthase (FAS) and the thermogenic marker UCP. IGF-I-induced differentiation-related gene expression at 24 h in both primary and immortalized brown adipocytes was mediated by an increase in p21ras.GTP active protein content. Transformed cell lines overexpressing exogenous p21ras (mainly in its ras.GTP active form) constitutively showed a higher lipid content and a higher FAS and UCP mRNA expression compared to primary and immortalized cells. These transformed cells were IGF-I independent with respect to their studied differentiation-related parameters. Additionally, transient transfection of primary brown adipocytes with the transforming ras gene induced UCP and FAS mRNA expression as well as cotransactivated UCP-chloramphenicol acetyltransferase fusion gene. Moreover, IGF-I transactivation of UCP promoter was partially precluded by cotransfection with the dominant-negative ras gene. Our results strongly suggest that IGF-I/p21ras induces adipogenic- and thermogenic-related gene expression in brown adipocytes.

Adipocytes↗
PubMed1996

Occupational allergy due to spider mites: Tetranychus urticae (Koch) and Panonychus citri (Koch).

BACKGROUND: Allergy to both house dust and storage mites is well established, but information about other species of mites is scant. OBJECTIVE: One hundred and fifty patients directly exposed to an occupational environment were studied to assess whether spider mites (Tetranychidae) caused their allergic symptoms. We also studied a group of 50 patients from an urban environment, who were not occupationally exposed to spider mites, with a strong sensitization to Dermatophagoides pteronyssinus (RAST class 4). METHODS: Case history (including questions about work-related symptoms), skin tests, RAST and conjunctival provocation tests were performed in both groups using Tetranychus urticae and Panonychus citri extracts as allergens. Cross-reactivity between spider mites and D. Pteronyssinus was determined by RAST inhibition. RESULTS: Fifty-four of 150 rural workers were positive to Tetranychidae and in all cases there was an associated sensitization to D. pteronyssinus. All individuals belonging to the urban group were positive to spider mites. RAST inhibition demonstrated a significant cross-reactivity between Tetranychidae and D. pteronyssinus. Five of fifty-four rural workers sensitized to spider mites developed symptoms only when they handled plants or fruits infested with spider mites and they became asymptomatic when exposure ceased. CONCLUSION: In the rural population studied, 36% of workers were found to be sensitized to spider mites and 10% had symptoms associated with occupational exposure. Since specific IgE antibodies to spider mites could not be detected in the absence of the specific IgE antibodies to D. pteronyssinus, and as all the affected workers were RAST positive to D. pteronyssinus, prior sensitization to house dust mites may be a risk factor for occupational allergy to spider mites.

Adult↗
PubMed1995

Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I.

Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation.

Adipocytes↗
PubMed1995

Transforming growth factor beta 1 induces differentiation-specific gene expression in fetal rat brown adipocytes.

Fetal rat brown adipocytes show a low number of transforming growth factor beta 1 (TGF-beta 1) binding sites of high affinity, revealing the presence of type I, II and III TGF-beta 1 receptors and a minor-labeled species of approximately 140 kDa. The culture of cells in the presence of TGF-beta 1 induced the expression of the tissue-specific gene uncoupling protein in a dose- and time-dependent manner. In addition, TGF-beta 1 up-regulates the expression of genes involved in adipogenesis such as fatty acid synthase, glycerol 3-phosphate dehydrogenase, malic enzyme and glucose 6-phosphate dehydrogenase, as well as induces the expression of fibronectin (specific target gene for TGF-beta 1). Our results suggest that TGF-beta 1 is a major signal involved in initiating and/or maintaining the thermogenic and adipogenic differentiation of rat fetal brown adipocytes.

Adipose Tissue, Brown↗
PubMed1995

Transforming growth factor beta modulates growth and differentiation of fetal hepatocytes in primary culture.

Fetal hepatocytes in primary culture are cells capable to carry out both proliferation and differentiation processes simultaneously. Previous studies have shown that these cells respond to mitogens, such as hepatocyte growth factor (HGF) or epidermal growth factor (EGF), inducing the expression of early genes, such as fos and myc. The transforming growth factor-beta (TGF-beta) family is one of the most influential groups of growth and differentiation factors. In this report, we show that TGF-beta 1 inhibits fetal hepatocyte proliferation, arresting these cells at G1 phase of the cell cycle. In addition, TGF-beta down-regulates the mitogen-induced myc early expression. However, TGF-beta has no effect on the expression of other protooncogenes, such as fos and H-ras. In addition to its inhibitory role on fetal hepatocyte growth, TGF-beta increases the mRNA levels of fibronectin, an extracellular matrix protein, and maintains the expression of some liver specific genes, such as albumin and alfafetoprotein, above control values. The analysis of the expression of some hepatocyte transcriptional factors has shown that TGF-beta increases HNF1 alpha and HNF1 beta mRNA levels. We conclude that TGF-beta may modulate liver growth and differentiation throughout fetal development.

Animals↗
PubMed1995

cAMP inhibits IGF-I-induced mitogenesis in fetal rat brown adipocytes: role of p21 ras.

Dibutyryl cAMP (DBcAMP) inhibited insulin-like growth factor I (IGF-I)-induced DNA synthesis of rat fetal brown adipocyte primary cultures. Its mitogenic inhibitory capacity, measured as [3H]thymidine incorporation into acid-precipitable material, was dose-dependent, being maximal at 0.5 mM. The entry of cells into S + G2/M phases of the cell cycle and the increase in cell number induced by IGF-I were partially inhibited by DBcAMP at 24 h and totally prevented at 48 h. DBcAMP inhibited the mRNA expression of c-myc induced by IGF-I at 2 h. Moreover, DBcAMP inhibited in parallel H-ras mRNA expression, p21 ras, and proliferating cellular nuclear antigen protein content induced by IGF-I at 24 and 48 h of culture, respectively, suggesting the involvement of p21 ras in the progression of fetal brown adipocytes through the S phase of the cell cycle and DNA synthesis.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Valvuloplasty in traumatic aortic insufficiency due to subtotal tear of the intima.

Aortic regurgitation is one of the usual pathologic findings necessitating valve replacement in cardiac surgery. Several diseases may result in leaflet incompetence. Circumferential intimal tear of the aortic root with prolapse of the aortic valve commissures is a rare cause of aortic incompetence. We report the repair of the aortic wall and valve in 1 patient with such a tear 6 months after an important thoracic trauma. Three months after the aortic valve reconstruction the patient is in good condition and fully asymptomatic.

Aged↗
PubMed1995

Regulation of gene expression by interleukin-6 in fetal rat hepatocyte primary cultures: role of epidermal growth factor and dexamethasone.

Fetal rat hepatocytes incubated in the absence of hormonal signals, or under proliferative (presence of epidermal growth factor [EGF]) or differentiative (presence of dexamethasone) culture conditions, showed responsiveness to interleukin-6 (IL-6). Northern blotting analysis for some typical acute phase genes such as haptoglobin and other proteins not previously identified as acute-phase reactants, such as alpha-fetoprotein, beta 2-microglobulin, and fibronectin, showed a positive modulation by IL-6, in a dose-dependent manner. However, a well-characterized negative acute-phase reactant such as albumin was not responsive to IL-6. The well-established synergism between glucocorticoids and IL-6 on inducing transcription is absent in fetal hepatocytes. Conversely, the combination of IL-6 and EGF produced different patterns of expression, depending on the messenger RNA (mRNA) analyzed. Thus, EGF abolished the increased mRNA levels of haptoglobin caused by IL-6 but had no effect on other genes such as alpha-fetoprotein and fibronectin.

Albumins↗
PubMed1994

Hepatocyte growth factor up-regulates met expression in rat fetal hepatocytes in primary culture.

Hepatocyte growth factor (HGF) is a potent mitogen for primary cultured fetal hepatocytes. In the present study, we have analyzed the c-met/HGF receptor expression in fetal hepatocytes and its modulation by growth factors and hormones. 20-day old fetal liver showed a barely expression of c-met mRNA levels. However, when fetal hepatocytes were incubated in the presence of HGF, a 10-fold increase in c-met mRNA levels was observed 30 min after addition of the factor. This HGF-induced effect on c-met expression was transient, losing its up-regulatory effect after 24 hours and returning to the initial levels at 48 hours. Transforming growth factor-beta, a negative regulator of fetal liver growth, increased c-met mRNA levels 48 hours after the addition of the factor, whereas glucocorticoids had a negative effect.

Animals↗
PubMed1994

IGF-I induces the uncoupling protein gene expression in fetal rat brown adipocyte primary cultures: role of C/EBP transcription factors.

Fetal rat brown adipocytes proliferate and reach confluence when cultured in the presence of serum, almost losing their expression of the tissue-specific gene uncoupling protein (UCP). Confluent cells in a serum-free medium induced the expression of UCP in response to 1.4 nM IGF-I at 72 h, in a time-dependent manner. This effect was not produced by 1 nM insulin. However, insulin but not IGF-I induced the expression of the lipogenic marker malic enzyme, suggesting that IGF-I but not insulin is involved in the thermogenic differentiation process of fetal brown adipocytes. Furthermore, the expression of C/EBP transcription factors was present in fetal brown adipocytes prior culture, decreasing during cell proliferation throughout culture. After confluence, insulin induced the expression of C/EBP alpha and delta, whereas the expression of C/EBP beta remained essentially unmodified. IGF-I induced C/EBP alpha but decreased C/EBP delta, this might indicate that both transcription factors play a reciprocal role in the expression of the tissue specific UCP gene.

Adipocytes↗
PubMed1994

Noradrenergic modulation of albumin expression in growth-stimulated adult rat hepatocytes in primary culture.

Serum albumin is the most abundant protein synthesized by liver cells, and its production is a reliable indicator of the differentiated state of hepatocytes. We have recently shown that fetal rat hepatocytes cultured under proliferative conditions, i.e., in the presence of EGF, responded to glucagon and noradrenaline increasing albumin protein and mRNA levels (de Juan et al., 1992. J. Cell. Physiol., 152:95-101). This effect was mimicked by agents that increase cyclic AMP levels. In this report, we show that in regenerating liver, noradrenaline modulation of albumin expression seems to be different. Hepatocytes from hepatectomized rats were cultured at low cell density and in the presence of EGF. Under these conditions, noradrenaline, which acted synergistically with EGF increasing DNA synthesis (de Juan et al., 1992. Exp. Cell. Res., 202:495-500), produced a decrease in albumin mRNA levels. This effect was dose-dependent, being maximum at 1 microM noradrenaline. Noradrenergic effect seemed to be mediated by alpha 1-receptors, because it was blocked by prazosin, but not by propranolol. Other Ca(2+)-increasing agents, as vasopressin, angiotensin II, or ATP, did not produce any effect. However, albumin mRNA levels decreased when the cells were incubated in the presence of tetradecanoyl phorbol-13-acetate (TPA). In addition, noradrenergic modulation of albumin expression was blocked by staurosporine, a protein kinase inhibitor with relative specificity for protein kinase C. Thus we can conclude that the role of noradrenaline on the regulation of liver growth and differentiation changes from fetal to adult life. This change is probably due to its action on different receptors: beta-receptors in fetal hepatocytes and alpha 1-receptors in the adult liver.

Adenosine Triphosphate↗
PubMed1994

Glucose-6-phosphate dehydrogenase gene expression in fetal hepatocyte primary cultures under nonproliferative and proliferative conditions.

The culture of fetal hepatocytes at high cell density for 64 h in medium supplemented with 5 mM glucose produced an induction of glucose-6-phosphate dehydrogenase (G6PD) mRNA in a time-dependent manner. Insulin and triiodothyronine (T3), separately, increase G6PD mRNA expression, producing an additive effect at 64 h when combined. Glucagon and, to a greater extent, dibutyryl-cAMP decreased the G6PD mRNA expression observed in the presence of 5 mM glucose and T3. Dexamethasone repressed the G6PD mRNA expression induced by glucose and insulin and decreased this expression when induced by T3, regardless of the presence of insulin. At low cell density, EGF in the presence of dexamethasone induced in parallel DNA synthesis, G6PD mRNA content, and specific activity, while EGF failed to increase these parameters at high cell density. In addition, G6PD expression in proliferative fetal hepatocytes was unresponsive to lipogenic hormones.

Animals↗
PubMed1994

Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression.

Fetal rat brown adipocytes at the beginning of culture showed a minimal T3 nuclear binding capacity and very low expression of the c-erbA genes, with low steady state levels of the mRNA forms beta-type and 5.5- and 2.6-kilobase (kb) alpha-types. The levels of these mRNA species increased after 7 days in culture in the presence of thyroid hormone-depleted serum; however, no significant increase in nuclear T3-binding capacity was observed. The addition of T3 incremented the abundance of the c-erbA beta-mRNA, induced the appearance of the 6.6-kb c-erbA alpha-mRNA and increased the nuclear T3-binding capacity by 30-fold. Parallel analysis of the uncoupling protein (UCP) mRNA, immunoreactive UCP, and functional UCP (detected by its ability to bind GDP) demonstrated that T3 induced the expression of the UCP gene in long term treated fetal brown adipocytes in culture. The presence of noradrenaline, a positive control for UCP expression, increased only the level of expression of the 5.5-kb c-erbA alpha-mRNA, but values for nuclear T3-binding capacity similar to those obtained in T3-treated cells were observed. Simultaneous addition of both hormones gave a pattern of expression of the c-erbA-mRNA forms similar to those obtained with these agents individually, and no further increment in their separate effects was observed on the nuclear T3-binding capacity and UCP expression.

Adipose Tissue, Brown↗
PubMed1994

Recurrent Trichosporon beigelii endocarditis after aortic valve replacement.

Fungal endocarditis of the bioprosthetic heart valve, implanted in a patient without evidence of impaired immunity, is reported. Clinical manifestations of endocarditis appeared 7 years after aortic valve replacement for rheumatic disease and included embolization into the popliteal artery. Trichosporon beigelii was isolated from the cultured fragments of the embolus. Two valve replacements were performed because of recurrent infection during the following 4 years. In spite of prolonged antifungal therapy, the patient died from multiorgan septic involvement.

Adult↗
PubMed1993

Regulation of malic enzyme gene expression by nutrients, hormones, and growth factors in fetal hepatocyte primary cultures.

The culture of fetal hepatocytes for 64 h in medium supplemented with 5 mM glucose, T3, insulin, and dexamethasone resulted in the coordinate precocious expression of malic enzyme mRNA, protein, and specific activity. T3 was the main inducer; meanwhile, insulin exerted a small synergistic effect when added with T3. Dexamethasone had a potentiation effect on the T3 response of malic enzyme mRNA expression regardless of the presence of insulin. This effect of dexamethasone on T3 response of malic enzyme mRNA expression was time (64 h) and glucose dependent. Glucagon, and to a greater degree dibutyryl-cAMP, repressed malic enzyme mRNA as well as protein expression by T3 and dexamethasone, in the absence of insulin. Glucose and other carbon sources such as lactate-pyruvate or dihydroxyacetone induced the abundance of malic enzyme mRNA in the absence of hormones. Insulin and T3 produced a high accumulation of malic enzyme mRNA in lactate-pyruvate medium, this effect being decreased by dexamethasone. EGF suppressed the induction produced by T3 and dexamethasone on malic enzyme mRNA, while the expression of beta-actin mRNA remained essentially unmodified.

Animal Nutritional Physiological Phenomena↗
PubMed1993

Establishment of permanent brown adipocyte cell lines achieved by transfection with SV40 large T antigen and ras genes.

We established multiple clonal cell lines by transfection of primary rat fetal brown adipocytes (BAT) with constitutive or inducible gene constructs of SV40 large T antigen (SVLTag) alone or in combination with transforming or normal ras genes. While primary BAT cell cultures stop growing and expressing uncoupling protein (UCP) after several passages in culture, our transfected cell lines could be propagated indefinitely and kept properties specific of BAT cells. Interestingly, cells transfected with ras genes alone did not survive as permanent cell lines. In contrast, cells transfected with SVLTag alone or in combination with various ras genes could readily be established as continuous cell lines. BAT cells transfected with SVLTag alone showed fibroblastic, flat morphology and clear contact inhibition of growth. In contrast, cells cotransfected with SVLTag and ras genes were more fusiform and possessed increased proliferative capacity. Thus, cells cotransfected with normal ras reached higher cell density at confluency, while cells cotransfected with transforming ras were clearly refractile, lost contact inhibition of growth, and presented a transformed phenotype. These results are consistent with the notion that ras genes are inefficient at overcoming senescence of primary BAT cells and can only confer improved proliferative ability to cells that have already been immortalized by a cooperating SVLTag gene. RNA analysis showed that most transfected cell lines expressed UCP along with the exogenously transfected genes. This demonstrated that genuine BAT cells, and not other possible contaminating cell lineages, gave rise to our selected cell lines. The variety of cell lines generated may correspond to various stages of progression through BAT development and therefore be useful to characterize signaling pathways and metabolic regulation of this particular tissue.

Adipose Tissue, Brown↗