Functional and morphological studies of mitochondria exposed to undecagold clusters: biologic surfaces labeling with gold clusters.

This study reports morphological and functional alterations observed in respiring isolated mitochondria when they are exposed to nonpenetrating, positive electrostatically charged synthetic undecagold clusters. Modification of the undecagold clusters positive charges change or prevent the functional effects and the binding to the outside surface of the mitochondria. The mitochondrial functional alterations are dependent on the oxidative phosphorylation capacity of the isolated organelles.

IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin.

Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability.

A 58-kDa resident protein of the cis Golgi cisterna is not terminally glycosylated.

A 58-kDa Golgi protein (gp58) was previously identified and found to be concentrated in cis Golgi cisternae in several cell types (Saraste, J., Palade, G.E.

Sulfate contributes to the negative charge of podocalyxin, the major sialoglycoprotein of the glomerular filtration slits.

Podocalyxin is the major sialoprotein of the rat glomerulus. Its function is to maintain the filtration slits of the glomerular epithelium open by virtue of its high net negative charge. We have used biosynthetic labeling and oligosaccharide analysis to characterize the anionic-charge-carrying moieties on this protein.

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization.

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A.

Mannose 6-phosphate receptors: potential mediators of germ cell-Sertoli cell interactions.

These studies have demonstrated that mouse pachytene spermatocytes, round spermatids, and Sertoli cells synthesize mannose 6-phosphate receptors and that the proportions of the CI- and CD-MPRs vary markedly between cell types. Isolated spermatogenic cells synthesize predominantly the CD-MPR and lower levels of the CI-MPR. In contrast, cultured Sertoli cells selectively synthesize the CI-MPR, even though transcripts for the CD-MPR have been detected in these cells.

beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum.

The post-translational processing of beta-glucuronidase in BW5147 mouse lymphoma cells is slow relative to other newly synthesized lysosomal enzymes. To characterize this slow maturation the acid hydrolase was immunoprecipitated from cells pulse-labeled with [2-3H]mannose. Radiolabeled beta-glucuronidase migrated as the precursor form of the enzyme for up to 4 h of chase, whereas another acid hydrolase, beta-galactosidase, was processed completely to its mature form within this same time period.

Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor.

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor.

Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines.

Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker.

A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency.

Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme.

Receptor-mediated endocytosis and differential synthesis of mannose 6-phosphate receptors in isolated spermatogenic and sertoli cells.

Mannose 6-phosphate (Man 6-P) receptors participate in the targeting of both newly synthesized and extracellular acid hydrolases to lysosomes, and also may mediate the effects of a number of growth factors. In this study, Man 6-P receptors were isolated from [35S]methionine-labeled germ cells and Sertoli cells by phosphomannan-Sepharose affinity chromatography and were analyzed by polyacrylamide gel electrophoresis and autoradiography. Mouse pachytene spermatocytes and round spermatids isolated by unit gravity sedimentation synthesized predominantly the 46,000 mol wt (Mr) cation-dependent Man 6-P receptor and only low levels of the 270,000 Mr cation-independent Man 6-P receptor.

Varicella-zoster virus glycoprotein oligosaccharides are phosphorylated during posttranslational maturation.

Varicella-zoster virus (VZV)-infected human embryonic lung fibroblasts (HELF) do not release infectious virions into their growth medium. Extracellular virions are pleomorphic, suggesting that they are partially degraded before their release from cells. To examine the intracellular pathway of viral maturation, [2-3H]mannose-labeled virus-encoded glycoproteins were isolated from VZV-infected HELF.

Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells.

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine.

Characterization of protein-linked glycoconjugates produced by identified neurons of Aplysia californica.

The biosynthetic capabilities of individual neurons of the abdominal ganglion of the marine mollusc Aplysia californica have been analyzed after intrasomatic injection of 3H-monosaccharides. Glycopeptides prepared from the metabolically labeled cells were fractionated using serial lectin affinity and gel filtration chromatography. The fractionation procedure yielded eight populations of glycopeptides, and comparison of two different neurons (R2 and R14) showed that the quantity of the individual species produced is cell-dependent.

Serum factors alter the extent of dephosphorylation of ligands endocytosed via the mannose 6-phosphate/insulin-like growth factor II receptor.

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated.

Mannose processing is an important determinant in the assembly of phosphorylated high mannose-type oligosaccharides.

Phosphorylation of the high mannose-type oligosaccharides attached to newly synthesized acid hydrolases occurs in two sequential steps within the endoplasmic reticulum and the Golgi apparatus, and the products generated at the two sites differ with respect to the location of the phosphorylated mannose residue. To investigate the mechanism of this two-step phosphorylation, biosynthesis of the Man-6-P recognition marker was studied in class E Thy-1- and J774 cells metabolically labeled with [2-3H]mannose. Class E Thy-1- cells produce truncated high mannose oligosaccharides that lack 4 mannose residues from the alpha 1,6-branch of the core beta-linked mannose residue; three of the missing residues are potential phosphorylation sites.

Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides.

Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region.

Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation.

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides.

Postendocytic maturation of acid hydrolases: evidence of prelysosomal processing.

The mannose 6-phosphate (Man 6-P) receptor operates to transport both endogenous newly synthesized acid hydrolases and extracellular enzymes to the lysosomal compartment. In a previous study (Gabel, C. A.

Posologic evaluation of clindamycin, using a canine model of posttraumatic osteomyelitis.

Posttraumatic osteomyelitis attributable to Staphylococcus aureus infection was experimentally induced in 30 dogs, after which the dogs were treated with clindamycin at various dosage regimens. Of the regimens evaluated, oral administration of 11 mg of clindamycin/kg of body weight twice daily for 28 days was the most effective treatment for the osteomyelitis.

Silicone oil with high specific gravity for intraocular use.

Silicone oil with a higher specific gravity than that of intraocular fluid or polydimethylsiloxane may have special indications in vitreoretinal surgery. Trifluorsiloxane is such a substance, and therefore its biological compatibility was investigated in rabbit eyes. It was found that this substance was clinically well tolerated within the observation time of up to 6 months, even if there was some neovascularisation from the inferior limbus.