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P Crespo

Publications and source records attributed to P Crespo.

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

Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway.

We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.

3T3 Cells↗
PubMed1994

Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits.

Mitogen-activated protein kinases, MAP kinases or ERKs (extracellular signal-regulated kinases) are rapidly stimulated by growth-promoting factors acting on a variety of cell-surface receptors. In turn, ERKs phosphorylate and regulate key intracellular enzymes and transcription factors involved in the control of cellular proliferation. The tyrosine-kinase class of growth-factor receptors transmits signals to ERKs in a multistep process that involves Ras and a limited number of defined molecules. In contrast, ERK activation by G-protein-coupled receptors is poorly understood, as is the role of ras in this signalling pathway. We have explored in COS-7 cells the mechanism of ERKs activation by m1 and m2 muscarinic receptors, typical examples of receptors coupled through Gq proteins to induce phosphatidylinositol hydrolysis and to G(i) proteins to inhibit adenylyl cyclase, respectively. Here we present evidence that ERK activation is mediated by beta gamma subunits of heterotrimeric G proteins acting on a ras-dependent pathway.

Adenylate Cyclase Toxin↗
PubMed1994

Apolipoprotein E expression in the cerebellum of normal and hypercholesterolemic rabbits.

We have studied the expression of apolipoprotein E (ApoE) mRNA in the cerebella of control and experimental rabbits fed with a cholesterol-rich diet for 8 weeks. Cholesterol-treated rabbits show a dramatic increase in serum cholesterol levels; however, no significant variations in the expression level of cerebellar ApoE mRNA were found in comparison to control rabbits. In addition, no differences were observed between control and hypercholesterolemic rabbits in the in situ hybridization pattern of ApoE mRNA on cerebellar cortex sections. ApoE mRNA was localized in astroglial processes associated with Purkinje cell bodies and dendrites, granule cell clusters, blood vessels and nerve fibers of the white matter. No expression of ApoE mRNA was observed in Purkinje and granule cell neurons. Polarized light examination of cryostat cerebellar sections revealed the absence of cholesterol-rich microglia/macrophage cells induced by the hypercholesterolemia. In this way, neither reactive microglial cells nor perivascular phagocytes were found by ultrastructural analysis in hypercholesterolemic conditions. The pattern of glial fibrillary acidic protein of the astroglial cells of the cerebellar cortex as well as their nuclear size were unchanged following cholesterol treatment, indicating the absence of astroglial activation induced by hypercholesterolemia. Our results suggest that cerebellar ApoE does not contribute to the general cholesterol homeostasis outside of the brain and supports the view that this cerebellar ApoE is involved in paracrine and autocrine functions particularly related with synapse turnover and membrane remodelling of astroglial cells.

Animals↗
PubMed1993

Induction of apolipoprotein E expression during erythroid differentiation of human K562 leukemia cells.

Apolipoprotein E (ApoE) is the only apolipoprotein that is expressed in extrahepatic tissues. ApoE expression was studied in leukemia K562 cells differentiated towards erythroid or myelomonocytic lineages. When K562 cells were differentiated into the erythroid lineage by addition either of 1-beta-D-arabinofuranosylcytosine or hydroxyurea, an increase in ApoE mRNA and protein was detected. A weaker ApoE induction was also observed during phorbol ester-induced myelomonocytic differentiation. Previous work has associated ApoE expression to monocytic differentiation. The findings reported here indicate that ApoE overexpression is not associated with a specific lineage in myeloid differentiation and that may play a role in erythroid differentiation.

Apolipoproteins E↗
PubMed1993

A tactile stimulator for studying motion processing in the somatic sensory system of primates.

A tactile stimulator was built for studying motion processing in the somatic sensory system of primates. This stimulator is used for assessing the responses of neurons of the somatic sensory system to stimuli moving in any traverse distance (range: 2-20 mm), with a variety of velocities (range: 4-120 mm/s), forces (range: 0-60 gf), and in any scanning direction. The stimulator is highly automated and can be used in combined psychophysical and neurophysiological studies in humans and in behaving monkeys.

Animals↗
PubMed1993

Representation of tactile signals in primate supplementary motor area.

1. We have studied the neuronal activity in the supplementary motor area (SMA) of two monkeys who categorized the speed of moving tactile stimuli delivered to the glabrous skin of the hand ipsilateral to the site of cortical recording and contralateral to the responding arm. 2. A large number of SMA neurons responded to the stimuli of all speeds (176 of 522) but only when those stimuli controlled behavior. 3. A second class of SMA neurons responded differentially in the categorization task (35 during the stimuli and 51 during the reaction time period) and predicted its outcome. 4. To dissociate the interrupt target switches presses from the tactile categorization responses, sixteen neurons, which responded to the stimuli in all speeds, and 11 neurons, which discharged differentially, were tested in a visual control task. None of these two classes of neurons responded in this situation. 5. It is concluded that the SMA ipsilateral to sensory input and contralateral to the responding arm is involved in the sensory decision process in this somesthetic categorization task.

Afferent Pathways↗
PubMed1993

Down-regulation of c-myc gene is not obligatory for growth inhibition and differentiation of human myeloid leukemia cells.

Suppression of c-myc expression is observed during induced differentiation of several myeloid cell lines and it has been attributed to the cell growth arrest that accompanies terminal differentiation. To dissect the role of c-Myc in the proliferation-differentiation switch we have studied c-myc expression in K562 cells exposed to several chemical agents. This model system allowed us to discriminate between the growth arrest and differentiation phenomena as well as the induction of differentiation along two different lineages (erythroid and myelomonocytic). Our results showed that c-myc expression did not significantly decrease when growth inhibition is reversible, either by treatment with a differentiating agent such as hydroxyurea (which induced erythroid differentiation) or by a non-differentiating agent such as interferon-alpha. In contrast, c-myc expression decreased when cells underwent terminal differentiation, either along the myelomonocytic (by 12-O-tetradecanoylphorbol-13-acetate) or erythroid (by 1-beta-D-arabinofuranosylcytosine) lineages. These results indicated that c-myc down-regulation is not obligatory for growth arrest and non-terminal differentiation of human myeloid cells. In contrast, c-myc down-regulation occurred in terminal differentiation, but induction of myelomonocytic differentiation resulted in a greater loss of c-myc mRNA than induction of erythroid differentiation.

Cell Differentiation↗
PubMed1992

Downregulation of hepatic albumin mRNA in response to induced hypercholesterolemia in rabbits.

Albumin gene expression was studied in rabbits fed on a cholesterol-rich diet for up to 16 weeks. Livers from experimental animals showed extensive lipid deposition. Hepatic albumin mRNA abundance decreased along the treatment to very low levels after 16 weeks. An 8-fold decrease in the rate of transcription of this gene was also detected. This downregulation of albumin gene expression cannot be attributed to a general impairment of RNA synthesis, as expression of other liver-specific and housekeeping genes did not vary significantly. There was a decrease in ascitic fluid albumin levels after 10 weeks, although serum albumin levels remained unchanged throughout the treatment. Our results are discussed in view of the relationship of albumin levels with hypercholesterolemia.

Animals↗
PubMed1992

Foam cells from aorta and spleen overexpress apolipoprotein E in the absence of hypercholesterolemia.

We have studied Apo E expression in atherosclerotic lesions and spleens in rabbits after a cholesterol-rich diet is discontinued and plasma cholesterol levels return to normal values. After 16 weeks, foam cells are still present in the atherosclerotic lesions and Apo E expression persists restricted solely to the lesion as ascertained by in situ RNA hybridization and northern blot. Apo E expression is induced in spleens of hypercholesterolemic rabbits. However Apo E mRNA levels decrease in this organ parallel to the disappearance of lipid loaded macrophages. These results of in vivo studies indicate that Apo E overexpression in foam cells does not depend on high serum cholesterol levels, but is a characteristic of macrophages that have acquired the foam cell phenotype.

Animals↗
PubMed1992

Hypercholesterolemia induces differential expression of rabbit apolipoprotein A and C genes.

We have compared steady-state mRNA levels of apolipoproteins AI, AII, AIV, CI, CII and CIII in liver and small intestine of rabbits fed on a cholesterol-rich diet for up to 16 weeks. Apolipoprotein (apo) AIV mRNA was detected in both liver and small intestine, while apo AII was not detected in either organ. Apo CI, apo CII and apo CIII were expressed only in liver and apo AI mRNA was detected only in small intestine. In small intestine, apo AIV and apo AI mRNA levels increased to a maximum at the 4th and 12th week of treatment, respectively. In liver there was a parallel increase in the mRNA levels of apo AIV, apo CI, apo CII and apo CIII, with maximum levels after 4 weeks of treatment. A 3-fold increase was found in apo CII and apo CIII hepatic transcription rates between hypercholesterolemic and control rabbits after 4 weeks of treatment, no longer detectable after 8 weeks. However, no changes were found in apo AIV and apo CI transcription rates. Changes in apolipoprotein mRNA levels were accompanied by changes in plasma lipoprotein levels. Overall, these changes correlate well with the variations detected in the expression of the different apolipoprotein genes. Our results indicate that dietary cholesterol plays an important role in the regulation of these genes and that this regulation is tissue dependent.

Animals↗
PubMed1990

Induction of apolipoprotein E gene expression in human and experimental atherosclerotic lesions.

Hypercholesterolemia and atherosclerosis were induced in New Zealand White rabbits by cholesterol feeding. Apolipoprotein E mRNA levels in livers were found to be slightly increased, as determined by Northern blots. Apolipoprotein E gene expression was dramatically induced in rabbit atherosclerotic aortas with respect to healthy aortas. However, apolipoprotein E mRNA levels in atherosclerotic aortas were low as compared with the hepatic mRNA levels of the same animals. Interestingly, we also found a significant increase in apolipoprotein E expression in human atheromata with respect to healthy aorta from the same individual. This is the first report on apo E gene induction in human atherosclerotic lesions.

Animals↗
PubMed1989

Analysis of IncF plasmids evolution: nucleotide sequence of an IncFIII replication region.

We determined the nucleotide sequence of RepFIII, the IncFIII replication region of the plasmid pSU316. Our data confirmed that RepFIII belongs to the RepFIIA family. The comparison of the nucleotide sequences from several RepFIIA-family plasmids revealed that pSU316 and R1 replicons are almost identical (96% similarity). Most of the differences between them are clustered in the incompatibility determinant. Analysis of the rest of the sequence suggested that the divergence between R1 and pSU316 replicons is very recent.

Base Sequence↗
PubMedDate not supplied

Otoconial mineralization in post-natal development.

Otoconial mineralization has been studied in the utricle of post-natal rats, i.e. from birth to the 20th day. Calcium microanalytically detected is seen to decrease progressively until, after 20 days, when the level is rather lower than at birth. In the adult animal the calcium value is much higher, which leads us to believe that there is an immediate calcium loss that is later recovered. Calcium in the peripheral areas of the macula is proportionately less than in the central part.

Age Factors↗
PubMedDate not supplied

[A validity study of continuous Doppler].

INTRODUCTION: Ultrasound is regularly used for the detection of carotid stenosis which can be treated surgically. This technique includes continuous Doppler, which together with study of blood flow in the orbit is very useful and should be used systematically, not only in certain cases. We present a study of the usefulness of this technique in our Cerebral Vascular Pathology Laboratory. PATIENTS AND METHODS: We selected 708 patients studied sequentially over 10 years with continuous Doppler and conventional angiography (gold standard). The data obtained independently with each technique were compared in order to establish the degree of concordance between them and the corresponding indicators of validity. Contingency tables were drawn up for different cut-off points corresponding to the different degrees of stenosis (50, 70 and 100%). The parameters for validity (sensitivity, specificity, predictive value and precision) were calculated in each case. RESULTS: The percentage coincidence of the two techniques was 91.95%. At the cut-off points there was a high degree of precision (> 94.35%). The specificity and negative predictive value of Doppler compared with angiography was over 95% and 96% respectively. CONCLUSIONS: The validity markers obtained in our laboratory support the technique using continuous Doppler as the ideal non-invasive diagnostic method for carotid stenosis and this means that its systematic use is to be recommended in patients with suspected extracranial carotid disease. Moreover, the low cost of the equipment used as compared with other more sophisticated techniques should lead to its more widespread use as a first-line diagnostic method in all Neurology departments.

Adult↗