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P Prehm

Publications and source records attributed to P Prehm.

At least 37 records · Page 2Linked to original sources
PubMed1993

Immunoelectrophoretic pattern of native mucosal intracellular glycoproteins of hog healthy and drug-intoxicated stomachs and of hog body fluids.

Naturally occurring glycoproteins have been extracted from fundic and antral mucosal tissue of the hog stomach by means of nondegrading techniques. Major and retarded glycoprotein fractions separated by gel filtration were further dissociated from appreciable amounts of noncovalently bound proteins by CsCl density gradient centrifugation. Antisera to glycoprotein fractions of fundic and antral regions of the stomach were prepared in rabbits. The major fractions from both gastric regions have similar molecular mass (approximately 2 x 10(6)), sedimentation coefficient (approximately 31.5 s), and specific viscosity (approximately 1.6). Purified fractions from each region were further separated into two subfractions by affinity chromatography on wheat germ lectin. Glycoprotein subfractions from antrum and fundus differ appreciably in their carbohydrate and amino acids content, share antigenic determinants, but do not cross-react with anti-hog serum protein antisera. Further diversity in native mucin glycoproteins was observed by the use of one-(D) and two-dimensional (2D) immunoelectrophoresis; subfractions that cross-react with specific anti-hog gastric glycoproteins were found to contain three or more components. D-Immunoelectrophoretic analyses demonstrated (1) in vivo degradation of glycoprotein components of the major fundic fraction isolated from mucosal tissue of alcohol/acetyl salicylate-intoxicated hog stomachs and (2) in vitro catabolism of major fundic glycoproteins by corresponding mitochondrial lysosomal (ML) acid hydrolases. Furthermore, 2D-immunoelectrophoretic analyses showed that (1) hog synovial fluid and plasma proteins have similar prosthetic moieties as either reacted with anti-hog serum proteins antisera. Nonetheless, locations, shapes, and staining intensities of the immunoprecipitate lines differed, which is indicative of different structures of the carbohydrate moieties of components of synovial fluid and plasma proteins, and (2) only a minor fraction of hog cerebrospinal fluid cross-reacted with anti-hog serum protein antisera. This is contrary to the generally accepted deduction based on high-resolution 2D-electrophoresis, indicative of different compositional patterns of plasma and cerebrospinal fluids.

Animals↗
PubMed1992

Immunohistochemical localization of hyaluronan synthase in cornea and conjunctive of cynomolgus monkey.

Distribution of hyaluronan synthase was investigated in cornea and conjunctiva of Cynomolgus monkeys (Macaca fascicularis) using polyclonal antibodies against the streptococcal enzyme. Strong immunoreaction was found in the cell membranes of the corneal endothelium, corneal epithelium, and most of the conjunctival epithelium. In the corneal epithelium all cells except the basal ones stained. In the conjunctiva all cylindrical cells stained, whereas among the goblet cells one type showed intense membrane staining, the other remained unstained. In the limbal portion of the conjunctival epithelium, which in many other respects differs morphologically and functionally from the remaining conjunctiva, all membranes of the different layers of the stratified epithelium except the most superficial ones, appeared unstained. Staining was also seen in all stromal fibroblasts and capillary endothelial cells.

Animals↗
PubMed1990

Release of hyaluronate from eukaryotic cells.

The mechanism of hyaluronate shedding from eukaryotic cell lines was analysed. All cell lines shed identical sizes of hyaluronate as were retained on the surface. They differed in the amount of hyaluronate synthesized and in the proportions of hyaluronate which were released and retained. A method was developed which could discriminate between shedding due to intramolecular degradation and that due to dissociation as intact macromolecules. This method was applied to B6 and SV3T3 cells in order to study the mechanism of hyaluronate release in more detail. The cells were pulse-labelled to form hyaluronate chains with labelled and unlabelled segments, and the sizes of labelled hyaluronate released into the medium during the pulse extension period were determined by gel filtration. B6 cells released identical sizes of hyaluronate at all labelled segment lengths, indicating that no intramolecular degradation occurred. When chain elongation was blocked by periodate-oxidized UDP-glucuronic acid, hyaluronate release was simultaneously inhibited. These results indicated that B6 cells dissociated hyaluronate as an intact macromolecule. In contrast, SV3T3 cells released hyaluronate of varying molecular mass distributions during extension of the labelled segment, suggesting partial degradation. Exogenous hyaluronate added to SV3T3 cultures was also degraded. This degradation could be prevented by the presence of radical scavengers such as superoxide dismutase and tocopherol. Degradation of endogenous hyaluronate could be inhibited by salicylate. These results led to the conclusion that SV3T3 cells released hyaluronate not only by dissociation, but also by radical-induced degradation.

Cell Line↗
PubMed1990

Shedding of hyaluronate synthase from streptococci.

Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state.

Blotting, Western↗
PubMed1989

Identification and regulation of the eukaryotic hyaluronate synthase.

Hyaluronate synthesis is required for fibroblast detachment in mitosis and migration. It is regulated by the activity of the synthase which is localized at the inner side of plasma membranes. The synthase was identified as a 50 kDa protein by immunological cross-reaction with the streptococcal enzyme and by affinity labelling. Transformation of fibroblasts by Rous sarcoma virus activated the synthase by enhanced transcription and phosphorylation. The synthase was a natural target of pp60v-src kinase.

Affinity Labels↗
PubMed1986

Increased hyaluronate synthesis is required for fibroblast detachment and mitosis.

Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.

Cell Cycle↗
PubMed1986

Isolation of streptococcal hyaluronate synthase.

Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.

Bacterial Proteins↗
PubMed1985

Inhibition of hyaluronate synthesis.

UDP-GlcNAc (UDP-N-acetylglucosamine) and UDP-GlcA (UDP-glucuronic acid) were oxidized by periodate in the ribose ring and utilized as inhibitors for hyaluronate synthase in membrane fractions from the B6 cell line and in cell cultures of B6 cells and human fibrosarcoma HT 1080. Inhibition was irreversible and concentration-dependent and could be prevented in the case of periodate-oxidized UDP-GlcNAc by UDP-GlcNAc. Periodate-oxidized UDP-GlcNAc was shown to block chain elongation of hyaluronate. Introduction of periodate-oxidized UDP-GlcA into B6 cells by hypo-osmotic lysis of pinocytotic vesicles decreased hyaluronate synthesis by direct inhibition of the synthase. In HT 1080 cells the synthesis of hyaluronate, chondroitin sulphate and heparan sulphate was inhibited simultaneously.

Cell Line↗
PubMed1984

Hyaluronate is synthesized at plasma membranes.

The hybrid cell B6 line, which synthesizes large amounts of hyaluronate as the predominant glycosaminoglycan, was grown in the presence of [3H]glucosamine. The [3H]hyaluronate has a high molecular weight and was excluded by Sephacryl S-1000. After disruption of the cells the [3H]hyaluronate could further be elongated by incubation with UDP-GlcNAc and UDP-[14C]GlcA, yielding a hybrid molecule of hyaluronate labelled with [3H]GlcNAc and [14C]GlcA. Treatment of the cells with hyaluronidase before disruption eliminated the large [3H]hyaluronate and elongation of nascent chains in vitro commenced from low-molecular-weight chains. Thus nascent hyaluronate chains were degraded extracellularly by hyaluronidase and were therefore synthesized at the inner side of plasma membranes and extruded to the cell surface.

Cell Line↗
PubMed1984

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.

Coliphages↗
PubMed1983

Changes in proteoglycan composition of F9 teratocarcinoma cells upon differentiation.

Teratocarcinoma cells (F9) were induced to differentiate by retinoic acid and then labelled with [3H]-glucosamine and [35S]-sulphate. Proteoglycans were then isolated from the plasma membranes and the culture medium by mild, dissociative, non-shear-dependent techniques. The undifferentiated cells were devoid of hyaluronic acid and contained negligible quantities of heparan sulphate, dermatan sulphate and chondroitin sulphate. Upon differentiation, the cells synthesized large amounts of hyaluronic acid and there was a threefold increase in the amount of membrane-bound sulphated proteoglycans. The differentiated cells also synthesized a proteoglycan (PGM-2) which was shed completely into the medium. It consisted of a large protein core with covalently linked sugar chains which were sulphated and had an approximate molecular weight of 12000. These sugar chains consisted of glucosamine and galactose in a molar ratio of 1:1 and contained a small quantity of mannose. Upon differentiation of the cells the amount of this molecule increased by threefold. This molecule was distinct from other proteoglycans since it was resistant to degradation by heparanase, chondroitinases, hyaluronidase and neuraminidase, but could be degraded by keratanase. Structurally it was very similar to keratan sulphate, consisting of alternating residues of (-Gal-GlcNAc-) in chains of approximately 20 such disaccharide units.

Cell Differentiation↗
PubMed1983

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase.

Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.

Acetylglucosaminidase↗
PubMed1983

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth.

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.

Cell Membrane↗
PubMed1983

Glycosaminoglycan synthesis in skin fibroblasts from patients with osteogenesis imperfecta.

Glycosaminoglycans were analysed from skin fibroblasts with osteogenesis imperfecta (OI) IIA and IIB. The content of sulphated glycosaminoglycans was greatly increased over age-matched controls and to a lesser extent with respect to older age control. Dermatan sulphate in comparison with older control was unaltered in the cells of OI IIA and IIB. The concentration of heparan sulphate was higher in the cells than in the medium, whereas hyaluronic acid, chondroitin sulphate and dermatan sulphate content was higher in the medium. The level of hyaluronic acid was greatly elevated in the medium of OI IIB with respect to both controls.

Cells, Cultured↗
PubMed1982

Rates of synthesis of basement membrane proteins by differentiating teratocarcinoma stem cells and their modulation by hormones.

The embryonal carcinoma mouse cell line F-9 was used as a convenient model for a quantitative study of the production of the basement membrane proteins laminin and type IV collagen. Both proteins could be identified in the culture medium and cell layer by radioimmuno assays, metabolic labeling and immunofluorescence. More than 95% of the material is secreted into the medium. Lack of ascorbic acid inhibits secretion of type IV collagen but not of laminin. Induction of differentiation into endoderm-like cells by retinoic acid consistently caused after a lag period of 2-3 days a 5-10 fold increase in the production of basement membrane proteins but not of total protein. Dibutyryl cyclic AMP further potentiated this specific effect particularly with respect to type IV collagen synthesis. Insulin, epidermal growth factor and nerve growth factor produced only moderate increases (10-60%) in the amount of laminin and type IV collagen. Effects of these hormones were only observed with certain doses and were quite variable between different experiments.

Animals↗