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Kenji Okuda - One of the best experts on this subject based on the ideXlab platform.

Natalie S. Cohen - One of the best experts on this subject based on the ideXlab platform.

  • intracellular localization of the mrnas of Argininosuccinate synthetase and Argininosuccinate Lyase around liver mitochondria visualized by high resolution in situ reverse transcription polymerase chain reaction
    Journal of Cellular Biochemistry, 1996
    Co-Authors: Natalie S. Cohen
    Abstract:

    Argininosuccinate synthetase and Argininosuccinate Lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of Argininosuccinate synthetase and Argininosuccinate Lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/10 nm gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver “enrichment ratio” for each type of cell structure. These ratios showed that the mRNAs for Argininosuccinate synthetase and Argininosuccinate Lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of Argininosuccinate synthetase and Argininosuccinate Lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAS. © 1996 Wiley-Liss, Inc.

  • Intracellular localization of the mRNAs of Argininosuccinate synthetase and Argininosuccinate Lyase around liver mitochondria, visualized by high‐resolution in situ reverse transcription‐polymerase chain reaction
    Journal of cellular biochemistry, 1996
    Co-Authors: Natalie S. Cohen
    Abstract:

    Argininosuccinate synthetase and Argininosuccinate Lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of Argininosuccinate synthetase and Argininosuccinate Lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/10 nm gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver “enrichment ratio” for each type of cell structure. These ratios showed that the mRNAs for Argininosuccinate synthetase and Argininosuccinate Lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of Argininosuccinate synthetase and Argininosuccinate Lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAS. © 1996 Wiley-Liss, Inc.

  • Argininosuccinate synthetase and Argininosuccinate Lyase are localized around mitochondria: An immunocytochemical study
    Journal of cellular biochemistry, 1996
    Co-Authors: Natalie S. Cohen, Aileen M. Kuda
    Abstract:

    Argininosuccinate synthetase and Argininosuccinate Lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that Argininosuccinate synthetase and Argininosuccinate Lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the Argininosuccinate synthetase and Argininosuccinate Lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.

Susumu Kawamoto - One of the best experts on this subject based on the ideXlab platform.

Bernard Joris - One of the best experts on this subject based on the ideXlab platform.

  • Sequence analysis of the ARG7 gene of Schizosaccharomyces pombe coding for Argininosuccinate Lyase
    Current Genetics, 1991
    Co-Authors: Roland Loppes, Reiner Michels, Isabelle Decroupette, Bernard Joris
    Abstract:

    The complete nucleotide sequence of the ARG7 gene, coding for Argininosuccinate Lyase (EC 4.3.2.1), in the fission yeast ( Schizosaccharomyces pombe ) has been determined. It consists of an open reading frame of 461 codons. The deduced protein has a molecular weight of 51 200 Da. The gene is devoid of introns which is confirmed by the fact that it is expressed in Escherichia coli after spontaneous insertion of a bacterial sequence probably bearing a prokaryotic promoter. A perfect “TATA” box is found at-72 and the major transcription initiation site in Saccharomyces cerevisiae is located at-11 as shown by primer extension experiments. Comparison of the S. pombe Lyase with related proteins from other organisms reveals an important degree of conservation except in the carboxyterminal part of the polypeptide. Additionally, a deletion removing 66 amino acids of the carboxy terminus yields an enzyme exhibiting some biological activity. A unique 1500 b transcript was found in S. cerevisiae when the intact gene was present, but the deleted version of the gene gave rise to at least three transcripts of 1800, 2800 and 3900 b.

Katsuro Iwase - One of the best experts on this subject based on the ideXlab platform.

  • Hyperammonemia: regulation of Argininosuccinate synthetase and Argininosuccinate Lyase genes in aggregating cell cultures of fetal rat brain
    Neuroscience Letters, 1999
    Co-Authors: Olivier Braissant, Masaki Takiguchi, Katsuro Iwase, Paul Honegger, Marc Loup, Claude Bachmann
    Abstract:

    Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by Argininosuccinate synthetase (AS) and Argininosuccinate Lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

  • Cloning of cDNAs encoding Argininosuccinate Lyase and arginase from Rana catesbeiana liver and regulation of their mRNAs during spontaneous and thyroid hormone-induced metamorphosis
    Biochimica et biophysica acta, 1995
    Co-Authors: Katsuro Iwase, Kiyoshi Yamauchi, Katsutoshi Ishikawa
    Abstract:

    Abstract Thyroid hormones are responsible for a change in the expression of many target genes during amphibian metamorphosis. In this study we cloned and sequenced cDNAs encoding two of the five urea cycle enzymes, Argininosuccinate Lyase and arginase, from adult liver of Rana catesbeiana. The cDNAs for the bullfrog Argininosuccinate Lyase and arginase encoded proteins of 467 and 321 amino acids with predicted molecular weights of 52 257 and 35 088, which were 72–75 and 64–68% identical to the mammalian enzymes, respectively. The accumulation of the mRNAs for Argininosuccinate Lyase and arginase in liver increased 26 and 4-times in a coordinated manner during spontaneous metamorphosis. Thyroid hormone-treatment induced about 5 and 10-times accumulation of mRNAs for Argininosuccinate Lyase and arginase in liver from premetamorphosing tadpoles within 4 days. These results suggest that the mRNA levels of the two enzymes in liver are upregulated by thyroid hormone during metamorphosis.