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E Roche

Publications and source records attributed to E Roche.

At least 55 records · Page 3Linked to original sources
PubMed1994

Calcium regulation of immediate-early response genes.

A rise in intracellular Ca2+ concentration induces the transcription of a number of eukaryotic genes through transcription factors interacting with calcium response elements. Immediate-early response genes encode proteins that couple extracellular signals to phenotypic alterations by modulating the transcription rates of target genes. Since the activation of early response genes occurs within minutes, this class of genes has served as a paradigm for the understanding of the molecular mechanisms by which external signals are conveyed to the nucleus to induce changes in genetic programs. In this review, we outline the recent information which has been gained specifically on how the Ca2+ messenger system modulates early response gene expression. We also discuss some lines of research with the intent of linking closer Ca2+ homeostasis and gene expression studies which in the past have followed their own separate routes.

Animals↗
PubMed1994

Role of oxygen free radicals in altitude-related disorders.

A massive expansion of mountain tourism and the practice of sports at altitude (mountaineering, skiing, cycling, hang-gliding, parapente, etc) has been observed in the last decades. This emphasis on new forms of sports and recreation represented as a social phenomenon is accompanied by an increase in the mountain associated-disorders and related-accidents. These include headache, lassitude, pain, difficulty in breathing, rapid heartbeat, and in the worst of the cases loss of consciousness, always possible, manifest by poor judgement, fatigue, etc, and death. However, medical studies are rare, mainly because the molecular mechanisms of tissue damage induced by oxygen free radicals are still poorly understood. Therefore, the goals of the present report are: 1) to summarize the main adaptations of the body at high altitude, introducing the concepts of altitude sickness and oxygen free radicals and their relation; 2) to propose a mechanism of action of oxygen free radicals in the development of this pathology, with special attention in hypoxia and related mechanisms; 3) to suggest a role for antioxidants in the therapy of altitude-related disorders.

Acclimatization↗
PubMed1994

Induction of c-fos in pituitary cells by thyrotrophin-releasing hormone and phorbol 12-myristate 13-acetate depends upon Ca2+ influx.

The role of cytosolic free Ca2+ ([Ca2+]i) in the induction of the immediate early gene c-fos by TRH or by phorbol 12-myristate 13-acetate (PMA) was studied in the clonal pituitary cell line GH4C1. It was found that c-fos mRNA levels were rapidly and transiently increased by TRH at physiological concentrations (1-100 nM). The effect of TRH was dependent on a rise in [Ca2+]i, and TRH stimulation of Ca2+ influx was essential for c-fos induction. Cell depolarization with K+, which produces a [Ca2+]i rise by soliciting Ca2+ influx via voltage-gated Ca2+ channels, was insufficient to induce c-fos. Blockade or downregulation of protein kinase C (PKC) strongly attenuated TRH stimulation of c-fos expression. Direct stimulation of PKC by PMA raised c-fos mRNA levels, but only under conditions permitting Ca2+ influx. We conclude that TRH induces c-fos mRNA by a mechanism dependent on PKC activation and on Ca2+ influx. The essential role of Ca2+ influx for PMA stimulation of c-fos mRNA suggests a novel pathway linking PKC stimulation to early gene expression.

Alkaloids↗
PubMed1993

Glucose regulates acetyl-CoA carboxylase gene expression in a pancreatic beta-cell line (INS-1).

Acetyl-CoA carboxylase (ACC) catalyzes the production of malonyl-CoA which may act as a metabolic coupling factor in nutrient-induced insulin release. We have studied the long term regulation of ACC by nutrients using the cell line INS-1. Glucose, from 5 to 20 mM, elicited a 15-fold increase in ACC mRNA. The effect was detected after 4 h and reached a maximum by 24 h. ACC protein accumulation followed that of ACC mRNA, and glucose did not modify the half-life of the ACC transcript. Glucose caused a dose-dependent rise in the glucose 6-phosphate content of INS-1 cells. 2-Deoxyglucose, which is phosphorylated by glucokinase but is not further metabolized, induced ACC mRNA. The effect of glucose was blocked by the glucokinase inhibitors mannoheptulose and glucosamine and was not mimicked by the 3-O-methyl or 6-deoxy analogues of glucose, which are not phosphorylated. Activation of the Ca2+, cAMP, and C-kinase pathways with high K+, forskolin, and phorbol 12-myristate 13 acetate, respectively, caused insulin release but not ACC mRNA induction. Basal insulin release, at 5 mM glucose, correlated with the ACC protein content of INS-1 cells preincubated for 24 h at various glucose concentrations. In conclusion, glucose is a potent inducer of the ACC gene, and glucose 6-phosphate may mediate its effect. Different signaling systems mediate the action of glucose on insulin release and ACC gene expression. The data strengthen the view that ACC plays a pivotal role in nutrient-induced insulin release.

Acetyl-CoA Carboxylase↗
PubMed1993

Intracellular Ca2+ and the regulation of early response gene expression in HL-60 myeloid leukemia cells.

To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration ([Ca2+]i) can be measured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel. Using promyelocytic HL-60 cells, we have observed an exquisite sensitivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since early and maximal inductions were observed at 200-300 nM [Ca2+]i. At early times (10-20 min), the [Ca2+]i dose dependence of c-fos transcription and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) [Ca2+]i. The threshold [Ca2+]i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of Ca2+ on a given gene can vary markedly as a function of both the [Ca2+]i concentration and the duration of stimulation. Strikingly, a [Ca2+]i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbation of [Ca2+]i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicated that Ca2+ promotes a small increase in transcriptional initiation and a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to [Ca2+]i, in terms of both the length of time and the dose of [Ca2+]i required for maximal gene induction, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c-fos intragenic element is the main target of Ca(2+)-regulated transcriptional activation.

Calcium↗
PubMed1993

Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes.

The molecular mechanisms involved in the degradation of individual cellular proteins are probably unique and characteristic. We have investigated in rat liver the degradation of glyceraldehyde-3-phosphate dehydrogenase, an abundant cytosolic enzyme of the glycolytic pathway. Immunoblot analysis of isolated liver lysosomes from rats treated with lysosomal inhibitors show that this protein is degraded, at least in part, by a lysosomal pathway. This pathway was further investigated by incubating the enzyme with lysosomes in a cell-free system, followed by proteolysis measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysosomes, and electron microscopic immunocytochemistry. We postulate that the degradative mechanism of glyceraldehyde-3-phosphate dehydrogenase includes a temperature-dependent lysosomal pathway, different from classical nonspecific macroautophagy. The postulated pathway involves: binding of the enzyme to the lysosomal membrane, entry into the lysosomal matrix, and degradation. This cell-free system, which can also incorporate in vitro synthesized proteins, should allow further advances toward clarifying the complex signals that regulate protein degradation as well as its close interrelationship with protein synthesis.

Animals↗
PubMed1993

Blockade of mevalonate production by lovastatin attenuates bombesin and vasopressin potentiation of nutrient-induced insulin secretion in HIT-T15 cells. Probable involvement of small GTP-binding proteins.

Small G-proteins (SMGs) require isoprenylation for their association with membranes. We have examined protein isoprenylation, subcellular distribution of SMGs, cytosolic Ca2+ changes and insulin secretion in HIT-T15 cells after treatment with lovastatin, which inhibits the production of isoprenoids by blocking mevalonate production by 3-hydroxy-3-methylglutaryl-CoA reductase. Numerous proteins in the 20-70 kDa range were found to be isoprenylated. Most of these proteins co-migrated with SMGs (21-27 kDa). Lovastatin treatment (25 microM, 24 h) decreased protein isoprenylation and affected the distribution of several SMGs, causing a large accumulation in the cytosol and a detectable decrease in membranes. Lovastatin selectively attenuated the potentiating action of bombesin and vasopressin, which activate phospholipase C in these cells, on insulin secretion stimulated by nutrients (glucose + leucine + glutamine). This lovastatin effect was overcome by mevalonate. Insulin secretion stimulated by nutrients alone or insulin release in the presence of the potentiating agents forskolin or phorbol myristate acetate remained unaffected. As the modulation of insulin secretion by isoprenaline and somatostatin were not altered by lovastatin, the drug does not non-selectively affect the binding of ligands to their receptors. Lovastatin did not interfere with the activation of phospholipase C by bombesin and vasopressin, since the rise in cytosolic Ca2+ induced by these agents was not changed. Limonene, proposed to block specifically prenyl-protein transferases of SMGs, did not alter protein isoprenylation patterns, but inhibited the stimulated insulin secretion. In conclusion, lovastatin selectively attenuated the potentiation of nutrient-induced insulin secretion by bombesin and vasopressin without affecting their activation of phospholipase C. The concomitant changes in SMG isoprenylation and their subcellular distribution after lovastatin treatment suggest that SMGs could play an important role in the bombesin and vasopressin action on insulin secretion.

Animals↗
PubMed1993

Oxidative stress in some dementia types.

By analogy to some pathologies (such as demyelinating diseases, arthritis and inflammatory processes) where the loss of cellular integrity is the starting point of tissue oxidative damage, it is proposed that some dementia types could be derived from a similar mechanism. The following oxidative events are proposed: (a) different agents could alter capillary or neuron integrity with the subsequent leakage of oxidases, proteases and transition metals from cellular compartments; (b) the persistence of the damaging agent, possible depletion of antioxidative defenses and concomitant loss of neuron function; (c) alteration of adjacent cells in the same manner; and (d) finally localized brain necrosis and progression of the dementia.

Alzheimer Disease↗
PubMed1989

ATP and 2,3-bisphosphoglycerate: models of metabolites for the regulation of intracellular protein degradation.

The main question in protein turnover is what determines the susceptibility of a given protein molecule to proteolytic degradation. Much evidence supports a role for the structural characteristics of individual proteins in determining their specific degradation rates. However, changes in the environment can influence these characteristics and thus the degradation rates. Since intracellular proteins in vivo are in a natural environment, substrates, products, cofactors and other low molecular weight compounds are often bound to the proteins, and probably contribute thereby to the vastly different half-lives of proteins. This paper reviews recent results from the authors' laboratory on the possible regulation of intracellular protein degradation by low molecular weight components. We have centered our studies on 2,3-bisphosphoglycerate and ATP, which modify, in opposite directions, the proteolytic susceptibility of specific mitochondrial and cytosolic proteins to lysosomal and non-lysosomal proteases. As shown, these metabolites can also modify the microautophagic uptake of certain proteins as well as the degradation rate of proteins in cultured cells.

2,3-Diphosphoglycerate↗
PubMed1988

The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins.

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.

Adenosine Triphosphate↗
PubMed1987

Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity.

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.

Alanine Transaminase↗
PubMed1987

Analysis by flow cytometry of rat hepatocytes from different acinar zones.

Many functional, morphological and biochemical differences among hepatocytes from different acinar zones have been described. Therefore, it will facilitate studies on liver metabolism rapid, non-destructive procedures to isolate hepatocytes from these zones. Flow cytometry is a new powerful tool which, however, has not been used thus far to accomplish the separation of hepatocytes from different acinar zones. We describe here various cytometric parameters which characterize hepatocyte populations, separated by isopycnic centrifugation in Percoll gradients. The intraacinar origin of the different hepatocytes was assessed by enzymatic and morphological measurements.

Alanine Transaminase↗
PubMed1987

2,3-Bisphosphoglycerate inhibits ATP-stimulated proteolysis.

Intracellular protein breakdown could be regulated at the substrate level by changes in the environment. Under in vitro conditions, ATP increases the proteolytic susceptibility of several mitochondrial and cytosolic proteins, while 2,3-bisphosphoglycerate not only has the opposite effect but also prevents the ATP-stimulated proteolysis. ATP and 2,3-bisphosphoglycerate, present at relatively high levels in many tissues, provide a good model of environmental components that may influence intracellular proteolysis.

2,3-Diphosphoglycerate↗
PubMed1987

2,3-Bisphosphoglycerate protects mitochondrial and cytosolic proteins from proteolytic inactivation.

2,3-bisphosphoglycerate at physiological concentration similar to that found in many tissues protects effectively ornithine transcarbamoylase (OTC) from proteolytic inactivation by broken lysosomes. 2,3-bisphosphoglycerate protects also many other mitochondrial and cytosolic proteins, such as glutamate dehydrogenase (GDH) an glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from proteolysis by broken lysosomes and other proteases. It is, thus, suggested that 2,3-bisphosphoglycerate may play an important role in the control of the degradative rates of some proteins, which may explain its high concentration in certain cells.

2,3-Diphosphoglycerate↗
PubMed1986

The reduction-oxidation status may influence the degradation of glyceraldehyde-3-phosphate dehydrogenase.

NADH and NADPH accelerate the 'in vitro' rate of proteolysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by elastase and other proteases, including lysosomal proteases. NAD+ and NADP+ have the opposite effect. Since there is a good correlation between proteolytic susceptibility of proteins and their 'in vivo' degradation rates, a possible role of the reduction-oxidation status in controlling the intracellular degradation of GAPDH is advanced.

Glyceraldehyde-3-Phosphate Dehydrogenases↗
PubMed1986

Regulatory mechanisms of intracellular proteolysis in mammalian cells.

Low molecular weight phosphoryl compounds, such as carbamoyl phosphate, 2,3-diphosphoglycerate and phytic acid protect, to different extents, mitochondrial and cytosolic proteins such as ornithine transcarbamoylase (OTC), carbamoyl phosphate synthetase (CPS), glutamate dehydrogenase (GDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), from proteolytic inactivation (rat liver lysosomal extracts, pronase, elastase). Given the wide variety and common occurrence of low molecular weight reagents such as typified here, it seems that this kind of inhibition may be important in the regulation of protein turnover. Regulation of intracellular proteolysis can also occur via the proteolytic systems. Immunocytochemical procedures for mitochondrial enzymes (CPS, GDH, OTC), show intracellular homogeneity, but intercellular heterogeneity in rat liver, compatible with a role of the autophagic-lysosomal system in degrading these proteins. However, degradation of short-lived proteins occurs by other mechanisms. Using centrifugation of cultured cells, we find that the Golgi apparatus takes part in the degradation of these proteins, probably by controlling the traffic of proteins or proteases to the degradation site.

Animals↗