Browsing by Author "SANTOS, MJ"

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    ANALYSIS OF PEROXISOMES IN LYMPHOBLASTS - ZELLWEGER SYNDROME AND A PATIENT WITH A DELETION IN CHROMOSOME-7
    (1993) SANTOS, MJ; MOSER, AB; DRWINGA, H; MOSER, HW; LAZAROW, PB
    Lymphoblasts are useful cells for the diagnosis and basic studies of several human genetic disorders. Peroxisomal disorders are usually diagnosed by using fibroblasts or blood samples. Here, we report the characterization of peroxisomes in lymphoblasts. We demonstrated that lymphoblasts from a patient with Zellweger syndrome, the prototypical disorder of peroxisome biogenesis, contained peroxisomal ghosts like those described previously in Zellweger fibroblasts. We also found that lymphoblasts that carry a deletion on chromosome 7 (q11.23q22.1), a region thought to contain one Zellweger syndrome gene, contained normal peroxisomes.
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    CHARACTERIZATION OF HUMAN PEROXISOMAL MEMBRANE-PROTEINS
    (1994) SANTOS, MJ; KAWADA, ME; ESPEEL, M; FIGUEROA, C; ALVAREZ, A; HIDALGO, U; METZ, C
    The peroxisomal membrane appears to play a crucial role in transporting proteins into the organelle. Some human genetic disorders involving peroxisome biogenesis, such as Zellweger syndrome, may be caused by genetic defects of the import machinery located in the peroxisomal membrane. In order to characterize the proteins of the human peroxisomal membrane, we isolated peroxisomes from human liver. We obtained their membranes using various procedures and analyzed their proteins by SDS-polyacrylamide gel electrophoresis and silver staining. We compared the protein composition of peroxisomal membranes with membranes derived from mitochondria and microsomes. The main peroxisomal membrane proteins (PMPs) have apparent molecular masses of 147, 112, 95, 87, 81, 79, 74, 69(70), 53-52 (double band), 47, 45, 43, 37, 31, 28, 22, and 17 kDa. The following PMPs of 147, 112, 79, 69(70), 53-52 (double band), 47, 43, 31, 28, 22, and 17 kDa fit the criteria for integral membrane proteins. We then produced rabbit polyclonal and mouse monoclonal antibodies that recognized some human PMPs. One of these antibodies detected mainly PMP43. We used this antiserum to evaluate the presence and subcellular distribution of the PMP43 in fibroblasts derived from patients affected by Zellweger syndrome. These results represent new information about the protein composition of the human peroxisomal membrane and provide biological tools for further characterization of the human PMPs and their genes in normal and pathological conditions.
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    CHARACTERIZATION OF PEROXISOMES IN CHINESE-HAMSTER OVARY CELLS IN CULTURE
    (1985) SANTOS, MJ; GARRIDO, J; OLIVER, C; ROBBINS, AR; LEIGHTON, F
    In order to explore the potential value of Chinese hamster overay (CHO) cells for the isolation of peroxisomal mutants defective in the peroxisomal fatty acid oxidation system, some characteristics of their peroxisomes were studied. Catalase was detected biochemically and histochemically in peroxisome-like particles in cells or in subcellular fractions prepared by differential centrifugation or isopyknic equilibrium in Percoll or Metrizamide with catalase in the high density fraction of the isopyknic equilibrium gradients. By oxidation system, exhibited an unusually high specific activity, 2.46 .+-. 1.09 mU/mg protein, in CHO cell homogenates, a value comparable to that of rat liver. This enzyme copurifies with catalase in the high density fractions of the isopycnic equilibrium gradients. By analogy with other cell types and from the ultrastructural analysis, it is concluded that these enzymes are contained in peroxisomes. These findings support the value of CHO cells for studies of peroxisomal function and organization.
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    IMMUNOLOCALIZATION OF A 43 KDA PEROXISOMAL MEMBRANE-PROTEIN IN THE LIVER OF PATIENTS WITH GENERALIZED PEROXISOMAL DISORDERS
    (1995) ESPEEL, M; ROELS, F; GIROS, M; MANDEL, H; PELTIER, A; POGGI, F; POLLTHE, BT; SMEITINK, JAM; VANMALDERGEM, L; SANTOS, MJ
    The presence of peroxisomal membrane ghosts was examined in liver biopsies from eleven patients presenting the clinical and biochemical picture of a generalized peroxisomal disorder (Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and variants of these syndromes). A polyclonal antibody raised against the membrane of human liver peroxisomes and recognizing a 43 kDa peroxisomal membrane protein (PMP) was used. In human control liver the antibodies react in a distinct and specific way with the peroxisomal membrane.
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    ISOLATION OF PEROXISOMES FROM FROZEN HUMAN LIVER SAMPLES
    (1992) ALVAREZ, A; HIDALGO, U; KAWADA, ME; MUNIZAGA, A; ZUNIGA, A; IBANEZ, L; KOENIG, CS; SANTOS, MJ
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    PEROXISOMAL ORGANIZATION IN NORMAL AND CEREBROHEPATORENAL (ZELLWEGER) SYNDROME FIBROBLASTS
    (1985) SANTOS, MJ; OJEDA, JM; GARRIDO, J; LEIGHTON, F
    The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of .apprxeq. 0.1 .mu.m in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated.
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    THE BASOLATERAL DOMAIN OF THE HEPATOCYTE PLASMA-MEMBRANE BEARS RECEPTORS FOR THE CIRCUMSPOROZOITE PROTEIN OF PLASMODIUM-FALCIPARUM SPOROZOITES
    (1992) CERAMI, C; FREVERT, U; SINNIS, P; TAKACS, B; CLAVIJO, P; SANTOS, MJ; NUSSENZWEIG, V
    Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. We show here that recombinant Plasmodium falciparum circumsporozoite protein (CS) binds specifically to regions of the plasma membrane of hepatocytes exposed to circulating blood in the Disse space. No binding has been detected in other organs, or even in other regions of the hepatocyte membrane. The interaction of CS with hepatocytes, as well as sporozoite invasion of HepG2 cells, is inhibited by synthetic peptides representing the evolutionarily conserved region II of CS. We conclude that region II is a sporozoite ligand for hepatocyte receptors localized to the basolateral domain of the plasma membrane. Our findings provide a rational explanation for the target cell specificity of malaria sporozoites.