Biomedical subjects
D Doyle
Publications and source records attributed to D Doyle.
Electron microscopy of gastric mucosal innervation in rats.
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Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes.
The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.
Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver.
RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.
Chronic cerebellar stimulation in cerebral palsy.
This report summarizes the experience with chronic cerebellar stimulation in cerebral palsy patients at the Children's Hospital of Eastern Ontario. From July, 1977, to September, 1978, 12 patients suffering from cerebral palsy underwent cerebellar implant for chronic cerebellar stimulation. Postoperative evaluation was made approximately 1 week, 3 months, 6 months, and 12 months after surgery by a group of specialists using speech, respiration, muscle tone, involuntary movements, salivation, and activities of daily living as parameters, and these findings were compared with the findings prior to surgery. Chronic cerebellar stimulation did not noticeably alleviate symptoms and signs of cerebral palsy nor did it improve activities of daily living in a significant number of patients.
Learning about death: a project report from the Edinburgh University Medical School.
A report of a problem-based learning project on the ethics of terminal care, offered as one of the options available to first year MB ChB students in Edinburgh University Medical School. The project formed part of the 'clinical correlation course' in the new curriculum. Six students took part under the supervision of two clinical tutors and a moral philosopher. The course was case-based and practical with students being given the opportunity over a period of eight weeks to meet patients, relatives and hospital staff at a local geriatrics hospital and terminal care home. The main issue studied was the degree of choice available to patients electing to be treated at home, in hospital or in a hospice. Other issues included: pre-death, disposal of the dead, certification of death, communication with relatives and follow-up bereavement services.
Metabolic effects of total parenteral nutrition with or without fructose, with special reference to the liver adenine nucleotide content.
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Arthritis: anti-inflammatory drugs in practice.
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Insertion of biologically active membrane proteins from rat liver into the plasma membrane of mouse fibroblasts.
Phospholipids, glucolipids, and total proteins were separated from a plasma membrane fraction of rat liver. Membrane glycoproteins were isolated from deoxycholate extracts of rat liver membranes and hepatoma tissue culture membranes by concanavalin A chromatography. The membrane glycoprotein on hepatocytes that acts as a receptor for serum glycoproteins have lost their terminal sialic acid was also purified from rat liver membranes. Closed membrane vesicles were reconstituted from mixtures of deoxycholate-solubilized phospholipids and proteins by dialysis and purified by isopycnic centrifugation. The orientation of the proteins and glycoproteins in these reconstituted vesicles was examined by their accessibility to trypsin and neuraminidase and by their ability to be released from the vesicle by different concentrations of detergent. Most of the proteins are embedded in a right-side-out orientation in the lipid bilayer. The reconstituted membrane vesicles can be fused to mouse L-cells with polyethylene glycol. The extent of fusion is a function of the phospholipid:protein ratio in the reconstituted vesicles. After fusion, the phospholipid component of the vesicles mixes relatively rapidly with cell membrane lipids as judged by the immunofluorescence pattern of cells fused with lipid vesicles containing trinitrophenylated lipids. In contrast, proteins transferred to L-cells show restricted diffusion as judged again by immunofluorescence techniques. The metabolic turnover of proteins and glycoproteins after transfer to the plasma membranes of mouse L-cells was examined by radioisotopic methods. Total rat liver membrane proteins are very stable after transfer to the L-cells. Some of these proteins may be involved in the formation of an exoskeleton at the cell surface. Hepatoma tissue culture cell glycoproteins after transfer to the L-cells are less stable in terms of turnover properties than are total liver membrane proteins. However, some of these proteins are released into the medium as large molecular weight material rather than being degraded to small molecular weight, acid-soluble component. The receptor for serum asialoglycoproteins is relatively stable after transfer in reconstituted vesicles to the membrane of L-cells. Most of this hepatocyte-specific membrane glycoprotein is degraded to acid-soluble material with a half-life in the L-cell of at least 50 h. Transfer of the purified receptor in reconstituted vesicles to L-cells confers upon the recipient cell the biological activities specified and initiated by these receptors in hepatocytes.
Metabolic fate of cell surface glycoproteins during immunoglobulin-induced internalization.
Goat antibodies directed against a subset of the externally oriented plasma membrane glycoproteins of hepatoma tissue culture (HTC) cells were used to follow the metabolic fate of the membrane antigens and the specifically bound immunoglobulin molecules in this cell type in cultures. Analyses of the immunoprecipitates from cells labeled in situ with neuraminidase and galactose oxidase, followed by reduction with tritiated sodium borohydride, indicate that about 40% of the galactose-labeled plasma membrane glycoproteins are recognized by the antiserum. Fluorescent microscopic analyses of cells treated with fluorescein-conjugated immunoglobulins and analyses of trypsin accessibility indicate that probably all of the antibodies bound to the cell surface are patched and internalized within about 4 hr when the cells are subsequently cultured at 37 degrees C in the presence of rabbit anti-goat immunoglobulins. At the same time, the antigens are also interiorized. Analyses of the cellular localization of the interiorized antigens and antibodies by cell fractionation on Percoll gradients show that the immunoglobulins to the cell surface antigens and the antigens themselves migrate to the same region of the Percoll gradient as lysosomal hydrolases. Although the antibodies bind to the cell surface glycoproteins and bring about patching and interiorization, there is no effect on the degradation of the plasma membrane antigens labeled via the galactose oxidase/borohydride reduction method. Furthermore, the iodinated antibodies directed against these membrane glycoproteins behave in their turnover properties like membrane antigens; the cell-bound specific immunoglobulins have the same half-life as the membrane glycoproteins. When the cells that had been reacted with the goat antibodies to membrane glycoprotein were cultured in the presence of rabbit anti-goat immunoglobulins, degradation of the former antibodies was effectively decreased. Similar results were obtained with concanavalin A and antibodies directed against this plant lectin.
Dexamethasone regulates the program of secretory glycoprotein synthesis in hepatoma tissue culture cells.
The secretory glycoproteins synthesized by hepatoma tissue culture (HTC) cells were resolved by two-dimensional polyacrylamide gel electrophoresis of media from cells that were grown in the presence of [(3)H]fucose. These cells synthesize and secrete a complex set of fucose-containing glycoproteins. These secretory glycoproteins are distinct from those glycoproteins present in the plasma membrane of HTC cells. Incubation of HTC cells with dexamethasone has a pronounced effect on the quality and quantity (denoted here as the program) of secretory protein synthesis, as assayed by the short-term incorporation of labeled mannose, fucose, or methionine. The synthesis of two mannose- and fucose- containing glycoprotein series, one of 50,000 mol wt and a more heterogeneous series with mol wt of 35,000-50,000, is increased to a high level by the hormone; conversely, the synthesis of other secretory proteins, particularly one with mol wt of 70,000, is decreased or stopped completely. The synthesis of some major secretory proteins is not affected by the hormone. Dexamethasone has less of an effect on the composition of either total cell membrane glycoprotein or plasma membrane glycoprotein. But there is a decrease in the synthesis of a major membrane glycoprotein series with mol wt of 140,000. These effects of dexamethasone are relatively specific to HTC cells. Neither Reuber H-35 cells nor primary cultures of rat hepatocytes show the same response to the steroid. Two variant HTC cell lines, which were selected for their resistance to dexamethasone inhibition of extracellular plasminogen activator activity, respond only partially to the steroid-induced regulation of the secretory and membrane glycoproteins.
Brain damage in fatal non-missile head injury.
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Light microscopic identification of vagal nerve innervation of the rat stomach using methylene blue.
Using a methylene blue staining techique, the innervation in the rat stomach has been studied. Several distinctive patterns of beaded and unbeaded nerves were seen. Axons in the muscle coat gave branches which ramified in connective tissue and were occasionally related to blood vessels. Further nerve fibres passed directly between the muscularis mucosae and muscle coat whilst others ran between the epithelium and muscularis mucosae. A few fine terminal axons were observed close to cells throughout the epithelial layer. This method of identifying nerves will be useful in studying the consequences of vagal denervation and reinnervation following vagotomy in man and animals.
[Current status of terminal care in England. 6. Conversation with D. Doyle. 2. Problem of communication in terminal care. Interview by T. Kashiwagi].
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[Hospice: reports on the practice of terminal care in England. 5. A conversation with Dr. D. Doyle. 1. Conditions necessary for terminal care. Interview by T Kashiwagi].
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Domiciliary terminal care.
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Transfer of the hepatocyte receptor for serum asialo-glycoproteins to the plasma membrane of a fibroblast. Acquisition of the hepatocyte receptor functions by mouse L-cells.
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Biochemical diagnosis of pralactinoma.
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