Browsing by Author "HOLUIGUE, L"

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    CIPROFIBRATE, A CARCINOGENIC PEROXISOME PROLIFERATOR, INCREASES THE PHOSPHORYLATION OF EPIDERMAL-GROWTH-FACTOR RECEPTOR IN ISOLATED RAT HEPATOCYTES
    (1993) ORELLANA, A; HOLUIGUE, L; HIDALGO, PC; FAUNDEZ, V; GONZALEZ, A; BRONFMAN, M
    Ciprofibrate, a hypolipidaemic drug with carcinogenic and peroxisome-proliferation effects in rat liver, was found to increase the phosphorylation of epidermal-growth-factor receptor in P-32-labeled isolated rat hepatocytes. This effect was suppressed by protein-kinase-C inhibitors, and was accompanied by an almost complete inhibition of the receptor autophosphorylation normally induced by its ligand. However, in vitro experiments showed that protein-kinase-C phosphorylation of purified epidermal-growth-factor receptor was activated by ciprofibroyl-CoA, the acyl-CoA derivative of the drug, but not by the unmodified drug. Neither compound affected the ligand induction of epidermal-growth-factor-receptor autophosphorylation in isolated liver membranes. These results suggest that metabolically produced ciprofibroyl-CoA in liver cells would activate protein-kinase-C and produce changes in epidermal-growth-factor-receptor function.
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    EPIDERMAL GROWTH-FACTOR RECEPTOR IN SYNAPTIC FRACTIONS OF THE RAT CENTRAL-NERVOUS-SYSTEM
    (1992) FAUNDEZ, V; KRAUSS, R; HOLUIGUE, L; GARRIDO, J; GONZALEZ, A
    Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity I-125-EGF binding sites (K(d), 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (K(d), 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDa EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.
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    TRANSCRIPTION INITIATION SITES FOR THE POTATO MITOCHONDRIAL GENE CODING FOR SUBUNIT-9 OF ATP SYNTHASE (ATP9)
    (1994) LIZAMA, L; HOLUIGUE, L; JORDANA, X
    The potato mitochondrial atp9 gene has a simple expression pattern. To determine where transcription initiates, primary mitochondrial RNAs were labeled by in vitro capping and hybridized to the 5' flanking sequences of the atp9 gene. A single transcription initiation region was identified. Primer extension and nuclease S1 protection analyses were used to precisely map the transcript 5' termini in this region. These results indicate that transcription initiates at 121-128 bp upstream of the atp9 open reading frame, in a sequence which does not present any homology with proposed consensus sequences for plant mitochondrial promoters. Nuclease S1 protection were also used to map 3' termini 67-71 nucleotides downstream of a putative single-stem loop structure.