Biomedical subjects

G Velasco

Publications and source records attributed to G Velasco.

At least 55 records · Page 3Linked to original sources

PubMed1995

Effects of anandamide on hepatic fatty acid metabolism.

Incubation of rat hepatocytes with anandamide (arachidonoylethanolamide) inhibited acetyl-CoA carboxylase activity and fatty acid synthesis de novo without affecting fatty acid synthase. This was concomitant to a decrease in the intracellular levels of malonyl-CoA. Likewise, anandamide depressed both cholesterol synthesis de novo and the incorporation of exogenous palmitate into triacylglycerols and phospholipids. On the other hand, anandamide stimulated in parallel both carnitine palmitoyltransferase I activity and ketogenesis from palmitate, though ketogenesis from octanoate was unaffected. The effects of anandamide on hepatic fatty acid synthesis and oxidation were: (a) mimicked by arachidonic acid, a product of anandamide breakdown by anandamide amidase; (b) prevented by phenylmethylsulfonyl fluoride, an inhibitor of anandamide amidase; and (c) not affected by bisindolylmaleimide, a specific inhibitor of protein kinase C. Furthermore, delta 9-tetrahydrocannabinol had no effect on any of the parameters determined, ruling out the possibility that the effects of anandamide on hepatic fatty acid metabolism are mediated by the peripheral cannabinoid receptor. The results thus indicate that anandamide might function as a carrier of arachidonic acid in the modulation of hepatic fatty metabolism.

Acetyl-CoA Carboxylase↗

PubMed1994

Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues.

A cDNA encoding a novel member of the cysteine proteinase family of proteins has been cloned from a human breast carcinoma cDNA library, by using a polymerase chain reaction-based cloning strategy. The isolated cDNA contains an open reading frame coding for a polypeptide of 321 amino acids that has been tentatively called cathepsin O. This protein presents all the structural features characteristic of the different cysteine proteinases identified to date, including the active site cysteine residue that is involved in covalent intermediate formation during peptide hydrolysis. The cathepsin O cDNA was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade the synthetic peptides benzyloxycarbonyl-Phe-Arg-7-amido-4- methylcoumarin and benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin widely used as substrates for cysteine proteinases. Cathepsin O proteolytic activity was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), an inhibitor of this subclass of proteolytic enzymes, thus providing additional evidence that the isolated cDNA codes for an authentic cysteine proteinase. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that cathepsin O is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in normal cellular protein degradation and turnover.

Amino Acid Sequence↗

PubMed1994

Inhibition of carnitine palmitoyltransferase I by hepatocyte swelling.

Incubation of hepatocytes under conditions known to increase their volume, i.e. with amino acids (glutamine, proline) or in hypo-osmotic medium, decreased carnitine palmitoyl-transferase I (CPT-I) activity. This effect of hepatocyte swelling was antagonized by okadaic acid and dibutyryl-cAMP. Physiological concentrations of glutamate inhibited CPT-I activity in digitonin-permeabilized hepatocytes but not in isolated mitochondria. Results suggest that the amino acid-induced inhibition of CPT-I shares a common mechanism with the amino acid-induced stimulation of acetyl-CoA carboxylase and glycogen synthase [(1993) Eur. J. Biochem. 217, 1083-1089].

Acetyl-CoA Carboxylase↗

PubMed1994

Structure and expression in breast tumors of human TIMP-3, a new member of the metalloproteinase inhibitor family.

A new member of the metalloproteinase inhibitor family of proteins has been cloned from a complementary DNA library derived from a human breast tumor. The isolated complementary DNA contains an open reading frame 633 base pairs long, encoding a polypeptide of 211 amino acids, which has been called tissue inhibitor of metalloproteinase 3 (TIMP-3). This protein displays low sequence similarity to the previously known human TIMPs but shows a high degree of similarity with chicken inhibitor of metalloproteinase 3, a recently described metalloproteinase inhibitor stimulated during oncogenic transformation of chicken fibroblasts and with the ability to promote some phenotypic properties of transformed cells. Northern blot analysis of RNA from human tissues revealed that the TIMP-3 gene is expressed in placenta and uterus but not in liver and ovary. In addition, TIMP-3 transcripts were detected in all breast carcinomas examined. On the basis of these expression data in breast tumors, together with its high degree of structural homology with chicken inhibitor of metalloproteinase 3, a possible role for human TIMP-3 in the regulation of connective tissue turnover and remodeling is proposed.

Amino Acid Sequence↗

PubMed1993

Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva.

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse-transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.

Amino Acid Sequence↗

PubMed1993

Role of the centromedian thalamic nucleus in the genesis, propagation and arrest of epileptic activity. An electrophysiological study in man.

Twenty-eight patients underwent electrical stimulation of the centromedian (CM) thalamic nucleus as a neuro-augmentative procedure to control intractable seizures of various types. To assess the correct placement of electrodes, electrical stimulation at 3 and 6 Hz, 1.0 msec and 600-2000 uA pulses in trains of 30-60 sec were used while the EEG was recorded by scalp electrodes. Spontaneous seizure activities of various types were simultaneously recorded. In cases of focal epilepsy depth electrodes were used in order to study the sequence of activation of different structures. Three Hz electrical stimulation induced spike-and-wave complexes accompanied by a typical absence. Eight Hz induced recruiting-like responses with ipsilateral predominance and no clinical manifestations. Interictal spike activity was recorded in the CM in cases of generalized tonic-clonic convulsions (GTCC) and ictal spike pattern in the CM preceded discharges in other areas. In atypical absences cortical and CM discharges occurred simultaneously. It is concluded that the CM participates in the onset of GTCC and typical absences, in the propagation of secondary GTCC but not in myoclonic convulsions.

Electric Stimulation↗

PubMed1993

A sequence variation in the human cystatin D gene resulting in an amino acid (Cys/Arg) polymorphism at the protein level.

A polymorphism in the coding region of the human cystatin D gene has been detected by direct sequencing of amplified DNA from different individuals. The variation, resulting from a T/C transition in exon 1 of the gene, causes an amino acid variation, Cys/Arg, at the protein level. An allele-specific oligonucleotide hybridization assay was developed and used to demonstrate this polymorphism in the population. the deduced frequencies were 0.55 and 0.45 for the Cys and Arg variant-encoding alleles, respectively.

Amino Acid Sequence↗

PubMed1993

Human Zn-alpha 2-glycoprotein: complete genomic sequence, identification of a related pseudogene and relationship to class I major histocompatibility complex genes.

The human gene (AZGP1) encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a protein present in several biological fluids and produced by a subtype of breast carcinomas, has been cloned and its complete nucleotide sequence determined. The gene spans over 9.7 kb, and its overall organization and nucleotide sequence are very similar to those of the first four exons of class I MHC genes. However, the Zn-alpha 2-gp gene differs from these genes in several significant ways. It lacks the coding information for the transmembrane and cytoplasmic domains typical of MHC genes, which is consistent with its presence as a soluble protein in different physiological and pathological fluids. In addition, it contains a high density of repetitive sequences, including Alu, MER, and MIR elements, which are not present at equivalent positions in class I MHC genes. Finally, its 5'-flanking region lacks the class I MHC regulatory complex and the interferon consensus sequence characteristic of class I MHC genes. These findings may explain the different expression pattern of Zn-alpha 2-gp and class I MHC genes in human tissues. Southern blot hybridization of DNA from several species with a cDNA probe indicated that Zn-alpha 2-gp genes are present in a wide variety of animal species, including monkey, rat, mouse, dog, cow, and rabbit. The human genome also contains a putative Zn-alpha 2-gp pseudogene that has been isolated and partially characterized. This pseudogene has an intron-exon organization identical to that of the functional gene, but it presents two deleterious mutations in the third exon that lead to the appearance of premature stop codons. Finally, considering the lack of polymorphism in the Zn-alpha 2-gp gene in comparison with MHC genes, putative roles for this human glycoprotein in the transport of nonpolymorphic substances or in intercellular recognition processes are proposed.

Amino Acid Sequence↗

PubMed1993

Localization of the human cystatin D gene (CST5) to chromosome 20p11.21 by in situ hybridization.

The gene coding for cystatin D (CST5), a cysteine proteinase inhibitor, was mapped by fluorescent in situ hybridization to human chromosome 20p11.21. This assignment, together with previous data on mapping of members of the cystatin gene family, indicates that cystatin family II genes are all clustered on the short arm of chromosome 20, whereas cystatin family I and III genes are located on the long arm of chromosome 3.

Chromosome Banding↗

PubMed1992

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.

Antibodies, Monoclonal↗

PubMed1991

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor.

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.

Amino Acid Sequence↗

PubMed1989

Characteristics and regulation of a high conductance anion channel in GBK kidney epithelial cells.

Single anion selective channels have been studied in membrane patches of GBK cultured epithelial cells from bovine kidney. High conductance anion channels are exclusively observed in excised patches after holding them at membrane potentials larger than +/- 30 mV for seconds or minutes. Once activated, the channels show a steep voltage dependence, complex gating properties and multiple conductance levels. The major unit conductance is 300 pS (+/- 28; n = 12) in symmetrical chloride. The discrimination among different anions and between anions and cations is poor. The activity of the channels remains unchanged after addition of 10 mM EGTA to the cytoplasmic face of the membrane, but it is irreversibly inhibited by application of 1 mM 4-acetamido-4'-isothiocyano-2,2'-disulphonic acid stilbene (SITS). Neither permeabilization of the cell membrane to Ca2+ using the Ca2+ ionophore A23187, treatments which increase the cyclic AMP content of the cells, nor hypoosmotic shocks, activate high conductance anion channels in cell-attached patches in which the activity of the channels is subsequently demonstrated upon patch excision. These results indicate that high conductance anion channels with the same characteristics as those reported in different cell types are present in GBK cells and suggest that the physiological role for these molecular entities may be different from those previously postulated.

Animals↗

PubMed1988

1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.

We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types.

2,3-Diphosphoglycerate↗

PubMed1987

Intestinal brush border membranes contain regulatory subunits of adenylyl cyclase.

Cholera toxin alters intestinal function by stimulation of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing) or adenylate cyclase, EC 4.6.1.1]. The mechanism of this activation is unknown and particularly puzzling because adenylyl cyclase is confined to the basal lateral membrane of enterocytes, whereas it is the brush border membrane that binds the toxin and contains proteins that undergo cholera toxin-catalyzed ADP ribosylation. It is shown that cholate extracts from cholera toxin-treated brush border membranes can efficiently reconstitute adenylyl cyclase activity in the guanine nucleotide-binding regulatory component (Gs)-deficient cyc- variant of the S49 mouse lymphoma cell line (cyc- cells lack the alpha subunit of Gs needed to activate the catalytic subunit of adenylyl cyclase). Moreover, NaF (in the presence of Al3+) and guanyl-5'-yl imidodiphosphate mediate strong activation of cyc- adenylyl cyclase provided the cholate extracts of brush border membranes are also present. Therefore, it appears that brush border membranes contain high levels of regulatory subunits of adenylyl cyclase in the absence of catalytic subunits. This represents a previously unrecognized feature of this transduction system that presumably plays an important role in the derangement of intestinal cell function by cholera toxin.

Adenosine Diphosphate Ribose↗

PubMed1986

Permeability properties of isolated enterocytes from rat small intestine.

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.

Animals↗

PubMed1986

Protein kinase C from small intestine epithelial cells.

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.

Animals↗

PubMed1986

Calcium uptake by intracellular compartments in permeabilised enterocytes. Effect of inositol 1,4,5 trisphosphate.

Treatment of rat small intestine with EDTA produced isolated enterocytes with plasma membranes which were permeable to small ions. When resuspended in a medium designed to resemble the intracellular medium, Ca2+ was accumulated into the cells. Both mitochondrial and a non-mitochondrial (presumably endoplasmic reticulum) compartments were responsible for sequestering the cation, as indicated by the effects of the mitochondrial inhibitors oligomycin and antimycin and of the Ca-ATPase inhibitor sodium orthovanadate assayed at low (0.9 microM) and high (12 microM) free Ca2+ concentrations. Addition of inositol (1,4,5) trisphosphate induced a rapid release of Ca2+ from the non mitochondrial compartment. The effect of inositol trisphosphate was concentration dependent and showed 50% of maximal release at 2 M. Neither cyclic AMP nor dibutyryl cyclic AMP caused release of Ca2+. These findings lend novel support to the possibility that Ca-mediated control of ionic transport in the small intestine is exerted through the phosphatidylinositol-protein kinase C transduction mechanism.

Animals↗

PubMed1986

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine.

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.

Acridine Orange↗