Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.     

Partial disassembly of peroxisomes

Rat liver peroxisomes were subjected to a variety of procedures intended to partially disassemble or damage them; the effects were analyzed by recentrifugation into sucrose gradients, enzyme analyses, electron microscopy, and SDS PAGE. Freezing and thawing or mild sonication released some matrix proteins and produced apparently intact peroxisomal "ghosts" with crystalloid cores and some fuzzy fibrillar content. Vigorous sonication broke open the peroxisomes but the membranes remained associated with cores and fibrillar and amorphous matrix material. The density of both ghosts and more severely damaged peroxisomes was approximately 1.23. Pyrophosphate (pH 9) treatment solubilized the fibrillar content, yielding ghosts that were empty except for cores. Some matrix proteins such as catalase and thiolase readily leak from peroxisomes. Other proteins were identified that remain in mechanically damaged peroxisomes but are neither core nor membrane proteins because they can be released by pyrophosphate treatment. These constitute a class of poorly soluble matrix proteins that appear to correspond to the fibrillar material observed morphologically. All of the peroxisomal beta-oxidation enzymes are located in the matrix, but they vary greatly in how easily they leak out. Palmitoyl coenzyme A synthetase is in the membrane, based on its co-distribution with the 22-kilodalton integral membrane polypeptide.     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.     

Rhizomelic Chondrodysplasia Punctata,a Peroxisomal Biogenesis Disorder Caused by Defects in Pex7p,a Peroxisomal Protein Import Receptor: A Minireview

Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease correspondingto complementation group 11 (CG 11), the second most common of the thirteen CGs of peroxisomalbiogenesis disorders (PBDs). RCDP is characterized by proximal limb shortening, severely disturbedendochondrial bone formation, and mental retardation, but there is an absence of the neuronal migrationdefect found in the other PBDs. Plasmalogen biosynthesis and phytanic acid oxidation are deficient, butvery long chain fatty acid (VLCFA) oxidation is normal. At the cellular level, RCDP is unique in thatthe biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybemore, peroxisomal matrix proteins fail to be imported from the cytosol. In this review, we discuss recentadvances in understanding RCDP, most prominently the cloning of the affected gene, PEX7,and identification of PEX7 mutations in RCDP patients. Human PEX7 wasidentified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, whichhad previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targetingsequences (PTS2). Normal human PEX7 expression rescues the cellular defects in culturedRCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients,including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsensemutation which results in loss of the carboxyterminal 32 amino acids. Identification of RCDP as a PTS2import disorder explains the observation that several, but not all, peroxisomal matrix proteins aremistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to bePTS2-targeted.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Peroxisomal ghosts are intracellular structures distinct from lysosomal compartments in Zellweger syndrome: a confocal laser scanning microscopy study

Peroxisome ghosts are aberrant peroxisomal structures found in cultured skin fibroblasts from patients affected by Zellweger Syndrome (ZS), a genetic disorder of peroxisomal assembly. They contain peroxisomal integral membrane proteins (PxIMPs) and they lack most of the matrix enzymes that should be inside the organelle (Santos et al., Science 239 (1988) 1536-1538). Considerable evidence indicates that these ghosts result from genetic defects in the cellular machinery for importing newly-synthesized peroxisomal proteins into the organelle. In contrast to these observations, (Heikoop et al., Eur. J. Cell Biol. 57 (1992) 165-171) report that in Zellweger Syndrome, peroxisomal membranes are located within lysosomes and/or contain lysosomal enzymes. We have undertaken a more detailed and systematic investigation of this matter, employing confocal laser scanning microscopy (CLSM). In fibroblasts derived from ZS patients belonging to different complementation groups, peroxisomes were labeled with antibodies against PxIMPs and lysosomes were labeled with an antibody against a lysosome associated membrane protein (LAMP-2) or with LysoTracker. The results unambiguously demonstrated no appreciable colocalization of PxIMPs and LAMPs (or LysoTracker), indicating that peroxisomal ghosts are distinct subcellular structures, occupying separate subcellular locations.