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Biomedical subjects

P R Fryer

Publications and source records attributed to P R Fryer.

31 records · Page 2Linked to original sources
PubMed1989

Desmoglein II-derived glycopeptides in human epidermis.

Antisera raised against a major 78 kD glycopeptide from pig epidermis were used to identify desmoglein II-derived glycopeptides in the conA-binding material isolated from human epidermis. In whole CaCl2-separated epidermis the antiserum recognized conA-binding components with apparent Mr of 115, 100, 82, 68, 50, 48, and 46 kD. The 82, 68, 48, and 46 kD immunoreactive bands were present in normal stratum corneum and plantar callus. Psoriatic scales contained significantly more of the 82 kD components and less of the 48 and 46 kD bands. Psoriatic scales also contained a major 50 kD conA-binding component unrelated to keratins or desmoglein II. Proteolytic peptide mapping showed that the major immunoreactive bands in normal stratum corneum and plantar callus were also chemically related. The 82 to 46 kD immunoreactive glycopeptides in plantar callus coincided with the major coomassie blue stained bands and were homogeneous on two-dimensional gels suggesting that this tissue may be a valuable source of human desmoglein II-derived glycopeptides. An antiserum directed against the electrophoretically co-purified 48/46 kD glycopeptides from plantar callus recognized the 82 to 46 kD bands in immunoblotting. In indirect immunofluorescence of frozen skin sections this antiserum stained the surface of epidermal cells in the spinous and granular layers of the tissue. In immunogold labeling of paraformaldehyde-fixed skin sections affinity-purified antibodies stained intact desmosomes in spinous and granular cells and desmosomal remnants in the stratum corneum. The results are consistent with our hypothesis that desmoglein II undergoes limited cleavage to stable fragments during terminal differentiation. Proteolytic degradation appears to be incomplete in psoriatic epidermis.

Concanavalin A↗
PubMed1989

Ultrastructural changes accompany inhibition of proteoglycan synthesis in chondrocytes by Cyclofenil diphenol.

Cyclofenil diphenol, a weak non-steroidal oestrogen, profoundly inhibits [35S]proteoglycan synthesis in cultures of Swarm chondrosarcoma chondrocytes under conditions in which protein synthesis is only marginally reduced. In the present experiments it was shown that after a 40-min treatment with Cyclofenil diphenol (90 micrograms ml-1) most of the normally abundant Golgi stacks in these cells disappeared and after 60 min they were absent. After 2-3 h treatment the cisternae of the endoplasmic reticulum (ER) were grossly distended and transformed into large ribosome-studded vesicles containing flocculent and filamentous material. These changes were dependent on the concentration of Cyclofenil and were fully reversible within 21 h of withdrawing the drug. The ultrastructural changes differed in some aspects if protein synthesis was blocked with cycloheximide for 15 min or 180 min before and during treatment with Cyclofenil. The Golgi disappeared but the ER cisternae, though distended, formed a continuous network and swollen ribosome-studded vesicles did not develop. However, non-membrane-bounded structures containing lipid droplets and material of low electron density developed in the cytoplasm under these conditions. The ultrastructural changes induced by Cyclofenil differ from those induced by monensin and diethylcarbamazine, suggesting that the drug acts at a different point in the secretory pathway for macromolecules.

Animals↗
PubMed1989

Red cell membrane protein distribution during malarial invasion.

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

Animals↗
PubMed1988

Type IX collagen: a possible function in articular cartilage.

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.

Animals↗
PubMed1987

Immunochemical studies on the synthesis and secretion of link protein and aggregating proteoglycan by chondrocytes.

Chondrocytes from pig laryngeal cartilage were maintained in culture, and the biosynthesis and secretion of link protein and proteoglycan were studied using immunochemical, biochemical and immunolocalisation techniques. In the presence of monensin there was a dose-dependent inhibition of link protein secretion which was very similar to that of aggregating proteoglycan, and suggested that they followed the same intracellular pathway during biosynthesis. In the presence of cycloheximide there was a similar dose-dependent inhibition of the secretion of both link protein and proteoglycan. Kinetics of secretion following inhibition of synthesis by cycloheximide showed that both proteins had similar intracellular pool sizes. Analysis of protein core and glycosaminoglycan biosynthesis showed that the time for synthesis and glycosylation of proteoglycan was 22 minutes, and this was quickly followed (within 6 minutes) by secretion. Intracellular electron microscopic immunolocalisation using protein A-gold showed link protein to be present in the Golgi cisternae and vesicles, and double-labelling experiments showed link protein only to be detected in vesicles that also labelled for proteoglycan protein core. When chondrocytes were maintained in monolayer culture for 10 days the rate of biosynthesis and secretion of proteoglycan increased although that of link protein remained constant. The control of their biosynthesis was thus shown to be independent. Within 4 hours of secretion a high proportion of link protein was incorporated into proteoglycan aggregates.

Animals↗
PubMed1987

Evidence that major 78-44-kD concanavalin A-binding glycopolypeptides in pig epidermis arise from the degradation of desmosomal glycoproteins during terminal differentiation.

The major concanavalin A (Con A)-binding component in urea/deoxycholate/mercaptoethanol extracts from pig ear epidermis had an apparent Mr of 78 kD. In indirect immunofluorescence affinity-purified polyclonal antibodies against this glycopolypeptide strongly stained the surface of suprabasal cells in the epidermis of pig and human skin. Immunocytochemical labeling with gold-labeled second antibody localized this staining to externally disposed, trypsin-sensitive components of desmosomes. Western blotting showed that the 78-kD glycopolypeptide was immunologically related to several other Con A-binding components in pig epidermis. Immunoreactive components with Mr of 115 and 100 kD were membrane-bound, appeared to be susceptible to trypsin in intact epidermis, and were absent from the stratum corneum. Immunoreactive components of lower Mr (78-44 kD) were not membrane-bound, were resistant to trypsin in intact tissue, and were present predominantly in the keratinized layers of pig epidermis. The 115-44-kD glycopolypeptides were also recognized by antisera raised against desmoglein II/desmocollin glycoproteins isolated from bovine spinous layer desmosomes. In addition, these antisera reacted with 120- and 105-kD bands that were apparently not recognized by the anti-78-kD glycopolypeptide antiserum in immunoblotting. In immune precipitation the anti-78-kD glycopolypeptide and antidesmoglein II/desmocollin antisera precipitated comparable amounts of the radioiodinated 78-44-kD components. Both antisera also precipitated the 120- and 105-kD components although the anti-78-kD glycopolypeptide serum was less effective. Little reaction with the 115- and 105-kD components was observed in immune precipitation with either serum. Proteolytic peptide mapping confirmed that the various immunoreactive glycopolypeptides were biochemically as well as immunologically related. The results suggest that terminal differentiation in pig epidermis is accompanied by the orderly degradation of desmoglein II/desmocollin glycoproteins resulting in the accumulation of 78-44-kD glycopolypeptides in the stratum corneum. These glycopolypeptides may represent functionally important nonmembranous domains of cell-adhesion molecules in desmosomes.

Animals↗
PubMed1986

Collagen and elastin changes in D-penicillamine-induced pseudoxanthoma elasticum-like skin.

A patient with cystinuria who was treated with large doses of D-penicillamine for 19 years developed skin abnormalities resembling those seen in pseudoxanthoma elasticum. Biochemical and histological examination of the dermis showed that the collagen content, the ratio of the major genetic forms of collagen and the distribution of collagen types was normal. Light microscopy demonstrated the presence of vastly increased amounts of elastin in the dermis, and the individual elastin fibres were shown by electron microscopy to be abnormal; chemical analysis showed the elastin to be poorly cross-linked. Some of the collagen also appeared structurally abnormal, and biochemically resembled that seen in the dermis of a young child with respect to cross-linking and hexosyl-lysine content. The therapy led to an increased deposition of collagen and elastin fibres which appeared abnormal, and resulted in an increase in total skin surface area. These data indicate that D-penicillamine was not fully effective in inhibiting collagen and elastin cross-linking, and appeared to prevent or inhibit the natural maturation process of the collagen.

Aged↗
PubMed1985

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin.

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [35S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.

Animals↗
PubMed1985

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Animals↗
PubMed1984

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.

Acrylic Resins↗
PubMed1983

Technical difficulties overcome in the use of Lowicryl 4KM EM embedding resin.

Several technical difficulties have been overcome in the use of Lowicryl 4KM resin. In order to embed and section tissue satisfactorily in the resin, it has been found necessary to thoroughly degass the resin before infiltration and polymerisation. After irradiation with UV light, the blocks are further polymerised by exposure to daylight for 2-3 weeks and then stored under partial vacuum over dessicant.

Acrylic Resins↗
PubMedDate not supplied

A study of the distribution of 7-ethoxycoumarin O-de-ethylase activity in human placental subcellular fractions.

Human placental homogenates from maternal smokers and non-smokers were fractionated using differential centrifugation techniques. Yields of the various subfractions were determined and their homogeneity assessed using electron microscopic procedures. The distribution and response of 7-ethoxycoumarin O-de-ethylase activity towards inhibition by dimethylsulphoxide, alpha-naphthoflavone and 9-hydroxyellipticine inhibitors in the placental subfractions were investigated. The low yield of microsomal protein obtained following differential centrifugation of placental homogenates (2.5 +/- 0.2 mg protein per g placenta) highlights the extremely refractory nature of human placental tissue towards homogenization. Enzymic studies showed that the majority (75 per cent) of the original O-de-ethylase activity in homogenates from smokers and non-smokers was to be found in the crude nuclear fraction. The 7-ethoxycoumarin O-de-ethylase activity present in both homogenate and crude nuclear preparations from a maternal smoker was found to be inhibited by both alpha-naphthoflavone and 9-hydroxyellipticine to a lesser extent than the O-de-ethylase activity which was present in both mitochondrial and microsomal fractions. While this observation suggests the existence of more than one induced O-de-ethylase activity in the human placenta, the possibility that such differences in inhibitory response may be due to other factors (e.g. inhibitor solubility effects) cannot be excluded. Studies using the above inhibitors also confirmed the results of earlier work by demonstrating that the O-de-ethylase activity in placental homogenates and subfractions from non-smokers is qualitatively different from the O-de-ethylase activities induced as a result of maternal smoking.

7-Alkoxycoumarin O-Dealkylase↗