Browsing by Author "NUNEZ, L"

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    BILIARY LIPID SECRETION - IMMUNOLOCALIZATION AND IDENTIFICATION OF A PROTEIN ASSOCIATED WITH LAMELLAR CHOLESTEROL CARRIERS IN SUPERSATURATED RAT AND HUMAN BILE
    (1993) RIGOTTI, A; NUNEZ, L; AMIGO, L; PUGLIELLI, L; GARRIDO, J; SANTOS, M; GONZALEZ, S; MINGRONE, G; GRECO, A; NERVI, F
    Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids.
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    CHOLESTEROL CRYSTALLIZATION-PROMOTING ACTIVITY OF AMINOPEPTIDASE-N ISOLATED FROM THE VESICULAR CARRIER OF BILIARY LIPIDS
    (WILEY, 1993) NUNEZ, L; AMIGO, L; RIGOTTI, A; PUGLIELLI, L; MINGRONE, G; GRECO, AV; NERVI, F
    Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 mug/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.
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    COMPARATIVE STUDIES ON GLUCOSE PHOSPHORYLATING ISOENZYMES FROM VERTEBRATES .8. IMMUNOCHEMICAL STUDIES ON MAMMALIAN HEXOKINASES-A
    (1985) NUNEZ, L; DEIOANNES, A; URETA, T
    1. An immune serum elicited in a rabbit by injection of homogeneous brain hexokinase A was shown to be specific for the antigen. Other rat hexokinase isoenzymes (kexokinases B, C or D) did not present cross-reaction when tested by immunoinhibition of enyzme activity, double immunodiffusion and immunoadsorbent columns. 2. The enzyme activity of hexokinase A from several mammals (rodents, lagomorphs, artiodactyls) was partially inhibited by the immune serum. In the case of the mouse enzyme, the amount of serum required to inhibit 50% of the activity was five-fold higher than in the case of the rat enzyme. Enzymes from cow or sheep brain were only marginally affected. 3. Hexokinases A isolated from various mammals, tested against the rat enzyme, showed faint lines of precipitation and marked spurs in double immunodiffusion plates even when enzymes from closely related rodents were analyzed. Immunoadsorbent columns, on the other hand, were able to retain most of the activity of hexokinases A from the mammals studied. 4. Micro-complement fixation tests showed that hexokinases A from mammals outside the Order Rodentia were only partially recognized by the anti-hexokinase Arat serum. 5. The results suggest that amino acid substitutions on the hexokinase A molecule have occurred at a rather fast rate.
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    HYPOLIPEMIC DRUGS ARE ACTIVATED TO ACYL-COA ESTERS IN ISOLATED RAT HEPATOCYTES - DETECTION OF DRUG ACTIVATION BY HUMAN LIVER HOMOGENATES AND BY HUMAN PLATELETS
    (1992) BRONFMAN, M; MORALES, MN; AMIGO, L; ORELLANA, A; NUNEZ, L; CARDENAS, L; HIDALGO, PC
    The formation of acyl-CoA esters of the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate and nafenopin was studied in isolated rat hepatocytes. The concentration of ciprofibroyl-CoA in the liver of ciprofibrate-treated rats was in the range of 10-30-mu-M. The three drugs formed acyl-CoA esters when incubated with isolated hepatocytes. Their formation was saturable and reached a plateau after 30 min incubation. Maximal intracellular concentrations of ciprofibroyl-CoA and clofibroyl-CoA (100-mu-M and 55-mu-M respectively) were attained at 0.5 mM of the free drugs in the incubation medium, whereas for nafenopin-CoA, the maximal intracellular concentration (9-mu-M) was reached at 1 mM-nafenopin. At low concentrations of the hypolipidaemic compounds in the incubation medium a significant proportion of the total intracellular drug was present as its acyl-CoA ester (25-35% for ciprofibrate). When isolated hepatocytes were incubated with a ciprofibrate concentration comparable with that observed in the blood of drug-treated rats (0.1 mM), ciprofibroyl-CoA attained an intracellular concentration similar to that previously observed in the liver of treated rats. The formation of ciprofibroyl-CoA by isolated rat hepatocytes was stimulated by the addition of carnitine and partially inhibited by the addition of palmitate. Further, it was shown that human liver homogenates synthesized ciprofibroyl-CoA at a rate similar to that observed for rat liver homogenates. Solubilized human platelets also formed ciprofibroyl-CoA, although at a rate two orders of magnitude lower than that of liver. The results support the view that acyl-CoA esters of hypolipidaemic peroxisome proliferators may be the pharmacologically active species of the drugs.
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    PROTECTIVE ROLE OF BILIARY CHOLESTEROL AND PHOSPHOLIPID LAMELLAE AGAINST BILE ACID-INDUCED CELL-DAMAGE
    (W B SAUNDERS CO-ELSEVIER INC, 1994) PUGLIELLI, L; AMIGO, L; ARRESE, M; NUNEZ, L; RIGOTTI, A; GARRIDO, J; GONZALEZ, S; MINGRONE, G; GRECO, AV; ACCATINO, L; NERVI, F