Biomedical subjects

M Benito

Publications and source records attributed to M Benito.

At least 109 records · Page 6Linked to original sources

PubMed1993

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes.

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF-I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early-response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H-ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.

Adipose Tissue, Brown↗

PubMed1993

Platelet derived growth factor/tyrosine kinase receptor mediated proliferation.

PDGF heterodimer of A and B chains, a complete mitogen for 3T3 mouse fibroblasts, exemplifies those growth factors interacting with membrane associated tyrosine kinase receptors. Its binding to the PDGF-receptors results in receptor dimerization and subsequent activation of tyrosine kinase activity in the cytoplasmic protein domain, autophosphorylation of the receptor being the first event in the transduction cascade. Before the ligand-receptor complex is internalized and degraded, receptor stimulation is transmitted to the general transduction network, in which several tyrosine kinase substrates are activated by phosphorylation and changes the cytoplasmic biochemistry. These changes include cytoplasmic alkalinization, increases in the intracellular concentration of cyclic-AMP and Ca2+ and activation of protein kinase C through the degradation of phosphoinositides. The known substrates recruited by the PDGF-receptor association are phosphatidylinositol-3'-kinase, ras-GTPase-activating protein, phospholipase C-gamma, serine-threonine kinase Raf-1 and src and src-related tyrosine kinases. Upon binding of PDGF to its receptor, transactivation of transcriptional and nuclear factors such as c-fos and c-myc genes and dephosphorylation of c-jun occurs, V-sis, the oncogen of the simian sarcoma virus (SSV), is highly homologous to the c-sis/PDGF-B gene that encodes the homodimer of the B-chain of the PDGF receptor. Cells transformed by SSV have been studied as a model system for the autocrine stimulation of the PDGF receptor.

Animals↗

PubMed1993

Cardiac myxomas: surgical treatment, long-term results and recurrence.

Long-term results of the surgical treatment of cardiac myxomas are not completely understood due to its recurrence. We review our experience in 27 operated cases with a follow-up to 22 years (mean 6.5 years), in order to throw light on results and review the problem of recurrence. One patient had a previous recurrent myxoma. At surgery we resect all the tumors and their attachment bases. We prefer a biatrial approach, ample resection and exploration of all cardiac chambers. Hospital mortality was 3.7% (1 case of associated aortic valve replacement) and late mortality 7%. Long-term results were satisfactory, without recurrences. The postoperative life expectancy of these patients seems similar to that of the normal population, except in cases of recurrence or associated valve replacement. Recurrence is very low (4.7% in 526 cases reported in the literature), except in the case of young patients and recurrent, familial, multiple or complex myxomas. The multigrowth potential of the tumor seems more important than an inadequate surgical resection.

Adult↗

PubMed1992

Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression.

Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.

Animals↗

PubMed1992

Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active Ras.GTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP), insulin-treated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin.

3T3 Cells↗

PubMed1992

Hormonal regulation of malic enzyme and glucose-6-phosphate-dehydrogenase expression in fetal brown-adipocyte primary cultures under non-proliferative conditions.

The expression of malic enzyme and glucose-6-phosphate (Glc6P) dehydrogenase was investigated in primary cultures of fetal brown adipocytes after the prolonged presence (6 d or 10 d) of various hormones under non-proliferative conditions. The presence of triiodothyronine for 6 d and 10 d resulted in maturation of the triiodothyronine regulatory mechanism of malic-enzyme expression at the mRNA level. However, triiodothyronine had no effect on Glc6P dehydrogenase expression. Insulin increased malic-enzyme and Glc6P dehydrogenase expression at the mRNA and protein level after 6 d and 10 d of culture. The joint presence of triiodothyronine and insulin produced an additive effect on malic-enzyme expression at the mRNA and protein level after 6 d and 10 d of culture, by two independent mechanisms. Noradrenaline prevented the effect at the protein level after 6 d, but not after 10 d, probably due to loss of the beta-adrenergic response of brown adipocytes after prolonged culture. Triiodothyronine overexpressed the Glc6P dehydrogenase mRNA induced by the presence of insulin at 6 d and 10 d of culture. There was no adrenergic regulation of Glc6P dehydrogenase expression in cultured fetal brown adipocytes, regardless of the time of culture.

Adipose Tissue, Brown↗

PubMed1992

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones.

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.

Animals↗

PubMed1992

Phosphoenolpyruvate carboxykinase and glucose-6-phosphate dehydrogenase expression in fetal hepatocyte primary cultures under proliferative conditions.

Fetal hepatocytes cultured for 64 h in the presence of glucagon and dexamethasone maintain a quiescent state, showing a low expression of glucose-6-phosphate dehydrogenase (G6PD) and a high induction of phosphoenolpyruvate carboxykinase (PEPCK). Under these culture conditions, the presence of EGF produced hepatocyte proliferation, with a concomitant increase of DNA synthesis, DNA content, and G6PD expression, meanwhile the expression of PEPCK was drastically reduced. The presence of forskolin plus IBMX nearly suppressed the increase in DNA synthesis and G6PD expression induced by EGF, showing a very high expression of PEPCK. Accordingly, it is possible to establish an inverse relation between G6PD, highly expressed in proliferating fetal hepatocytes, and PEPCK expression, highly expressed in quiescent fetal hepatocytes under specific hormonal stimulation.

Animals↗

PubMed1991

Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes.

Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I (IGF-1). Expression of transfected ras oncogenes led to differentiation of these cells into adipocytes in the absence of externally added insulin or IGF-I. Cells transfected with normal ras genes or the tyrosine kinase trk oncogene did not differentiate. Transfection with a dominant inhibitory ras mutant resulted in inhibition of differentiation. Exposure of untransfected 3T3-L1 cells to insulin stimulated formation of the active Ras.GTP complex. These observations indicate that Ras proteins participate in signal transduction pathways initiated by insulin and IGF-I in these cells.

Adipose Tissue↗

PubMed1991

Histomorphometric evaluation of intrahepatic changes following portal overflow in the rat.

Intrahepatic changes after an end-to-side renoportal arteriovenous shunt were studied in 60 rats. Portal and vena caval pressures did not increase from 3 weeks to 1 year. The number and diameters of the portal and hepatic vein lumens were determined through morphometric evaluation to calculate the relative areas. The proportion of area composed by the portal veins increased 3 weeks after shunt creation (P less than 0.001), reaching its highest level at 1 year (0.08% in control rats vs. 2.45% +/- 0.1% at 1 year). The proportionate area for hepatic veins also increased after 3 weeks (P less than 0.001), reaching its highest value by 12 weeks (0.49% in controls vs. 1.73% +/- 0.08% at 12 weeks). Our results suggest that there was a dramatic drop in the intrahepatic vascular resistance, especially at the portal bed, which may have compensated for the portal overflow. The theories regarding forward overflow compensatory mechanisms are discussed.

Adaptation, Physiological↗

PubMed1991

Proliferation of fetal brown adipocyte primary cultures: relationship with the genetic expression of glucose 6 phosphate dehydrogenase.

Fetal brown adipocyte primary cultures increase DNA synthesis; cell number; and DNA, RNA, and protein contents in response to 10% fetal calf serum, IGF-I, and EGF plus vasopressin plus bombesin when added for 64 h to quiescent cells. IGF-I is a complete growth factor in this system while EGF needs the presence of vasopressin plus bombesin for its maximal proliferative effects. These mitogens induce the genetic expression of G6P dehydrogenase, increasing its mRNA content as well as its specific activity and amount of immunoreactive protein. The presence of cAMP elevating agents prevents the stimulatory effect of EGF plus vasopressin plus bombesin on DNA synthesis, cell number, and DNA content as well as on the induction of G6P dehydrogenase expression. Thus, changes on the proliferative state of these cells are associated with the level of expression of G6P dehydrogenase.

Adipose Tissue, Brown↗

PubMed1991

The caudal gluteal vessels: a suitable recipient bundle for free bone grafting in the canine femur.

The caudal gluteal vascular bundle was analyzed in the dog to determine its suitability as a recipient area for free vascularized bone grafts in the femur. The anatomy of the caudal gluteal artery and vein, as well as the arterial blood flow, were studied. This bundle was also used to revascularize 50 free vascularized rib grafts for proximal and femoral shaft reconstruction. The pedicle was constant, long (6.12 +/- 1.85 cm), and easily accessible via a lateral approach. It was large enough (artery: 1.74 +/- 0.46 mm, vein: 2.27 +/- 0.69 mm) and had an adequate blood flow (99 +/- 8 ml/mn) for microvascular techniques. Due to the existence of three anastomotic vascular rings with both the superficial and deep femoral bundles, these vessels had a bidirectional blood flow. In all the free vascularized rib grafts the caudal gluteal vessels were distally based (reversed blood flow) and provided a sufficient blood supply to the grafted bone. These results suggest that the caudal gluteal vessels are excellent recipient vessels for research in the field of free vascularized bone grafts using the canine femur.

Animals↗

PubMed1990

Development of the uncoupling protein in the rat brown-adipose tissue during the perinatal period. Its relationship with the mitochondrial GDP-binding and GDP-sensitive ion permeabilities and respiration.

In this paper we report the development of the immunologically detected uncoupling protein (UCP) in brown adipose tissue during the perinatal period in the rat and its relationship with its functional activity expressed in terms of GDP-binding capacity, GDP-sensitive permeabilities and GDP-sensitive respiration. Immunologically detected UCP increased during the last 2 days of foetal life (under euthermic conditions) and after birth (after postnatal hypothermia) during the early suckling period, reaching its maximum value on day 10 after birth. This increase in UCP is accompanied by parallel increases in the GDP-binding capacity, GDP-sensitive permeabilities to protons and chloride ions and GDP-inhibitable respiration. During the suckling--weaning transition, there was a regression of the parameters related to the functional activity of the UCP (GDP-binding capacity and nucleotide-sensitive permeabilities and respiration) without changes in the immunologically detected UCP. These results suggest that the involution of this tissue in the rat commences in this period; the first parameters affected are those related to the functional activity of the UCP while the UCP is still present in its highest level. This seems to support the idea that, in this period of development of the rat, the UCP may exist in the brown fat mitochondria in a functional (unmasked) form and a non-functional (masked) form.

Adipose Tissue, Brown↗

PubMed1990

Motor reflex responses elicited by cutaneous stimulation in the regenerating nerve of man: axon reflex or ephaptic response?

In 57 of 60 nerves (29 median and 31 ulnar) sutured at the wrist, forearm and arm, we recorded motor responses in thenar or hypothenar muscles by electrical stimulation of the corresponding fingers. Recordings were made at different times during the process of regeneration, ranging from 3 months up to 11 years. The responses showed a constant shape and latency to every stimulation (simple or repetitive). The latency was shorter the more distal the level of injury and the greater the elapsed time from the reinnervation. The point of "reflexion" of the responses is at or very near the line of nerve suture. The electrophysiological behavior of the responses fits well with either the criterion of axon reflex or ephaptic response. We discuss both possibilities and conclude that it is not possible, with the electrophysiological technique that we used, to distinguish between an axon reflex and an ephaptic response.

Adolescent↗

PubMed1990

Revascularization of femoral head ischemic necrosis with vascularized bone graft: a CT scan experimental study.

An ischemic necrosis of the femoral head was induced in 15 mongrel adult dogs using the technique described by Gartsman et al. [10]. Five weeks later, a free vascularized rib graft was transferred into the previously induced ischemic femoral head. High resolution computed tomographic scanning was used to evaluate revascularization 4, 8 and 12 weeks after grafting. The femoral head exhibited new vessel formation throughout the study. Arterial terminal branches arising from the rib graft medullary and periosteal circulations extended beyond the rib graft, entered the head, and reached the subchondral plate. Even where the rib graft did not replenish the central core of the head, there was vascular supply from the grafted bone's vascular tree. These results suggest that a free vascularized bone graft is able to revascularize an experimentally induced ischemic femoral head necrosis.

Animals↗

PubMed1990

Role of free vascularized bone grafts in the experimentally-induced ischemic necrosis of the femoral head.

In 15 mongrel adult dogs, an ischemic necrosis of the femoral head was produced, following the technique described by Gartsman and colleagues. Five weeks later, a 6- to 8-cm long rib graft was harvested with its vascular bundle and transferred into the previously induced ischemic femoral head. Microsurgical anastomoses were performed for revascularizing the rib graft. Dogs were studied using scintigraphy, blood flow measurements, roentgenograms, angiograms, and histology at four, eight and 12 weeks after grafting. New bone and vascular formation was exhibited throughout the study, as demonstrated by a highly positive scintigraphic activity and the formation of numerous arterial terminal branches arising from the rib graft circulation which entered the femoral head and reached the subchondral plate. The formerly necrotic femoral head bone exhibited osteoblastic activity, viable osteocytes, and well-populated bone marrow. The rib graft was also incorporated into the femoral head. These results suggest that a vascularized bone graft is able to repair a femoral head necrosis, and may be considered a rational approach for clinical purposes.

Anastomosis, Surgical↗

PubMed1990

Inability of donor total body irradiation to prolong survival of vascularized bone allografts: experimental study in the rat.

At the present time, the toxic side effects of recipient immunosuppression cannot be justified for human non-vital organ transplantation. Total body irradiation has proven effective in ablating various bone-marrow-derived and endothelial immunocompetent cellular populations, which are responsible for immune rejection against donor tissues. Irradiation at a dose of 10 Gy was given to donor rats six days prior to heterotopic transplantation of vascularized bone allografts to host animals. Another group of recipient rats also received a short-term (sixth to fourteenth day after grafting), low dose of cyclosporine. Total body irradiation was able merely to delay rejection of grafts across a strong histocompatibility barrier for one to two weeks, when compared to nonirradiated allografts. The combination of donor irradiation plus cyclosporine did not delay the immune response, and the rejection score was similar to that observed for control allografts. Consequently, allograft viability was quickly impaired, leading to irreversible bone damage. This study suggest that 10 Gy of donor total body irradiation delivered six days prior to grafting cannot circumvent the immune rejection in a vascularized allograft of bone across a strong histocompatibility barrier.

Animals↗