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Biomedical subjects

R G Anderson

Publications and source records attributed to R G Anderson.

At least 127 records · Page 7Linked to original sources
PubMed1984

Increase in membrane cholesterol: a possible trigger for degradation of HMG CoA reductase and crystalloid endoplasmic reticulum in UT-1 cells.

The crystalloid endoplasmic reticulum (ER) houses large amounts of HMG CoA reductase, the rate-controlling enzyme in cholesterol synthesis. The crystalloid ER appears in UT-1 cells, a line of Chinese hamster ovary cells that has been chronically starved of cholesterol as a result of growth in the presence of compactin, an inhibitor of reductase. When cholesterol was provided to UT-1 cells in the form of low density lipoprotein (LDL), the reductase and crystalloid ER were destroyed. This destruction was preceded by an increase in the cholesterol content of crystalloid ER membranes, as judged by a 4- to 8-fold increase in their ability to form complexes with filipin, a cholesterol-binding compound that can be visualized in freeze-fracture electron micrographs. Filipin binding to other membranes was unchanged. Thus insertion of cholesterol into the crystalloid ER membrane may trigger the degradation of reductase and the membrane itself.

Animals↗
PubMed1984

Inflammatory effects of otic drops on the middle ear.

Combination topical otic preparations are used to treat many infections of the external and middle ears. Despite the presence of ototoxic drugs in a number of these drops and convincing evidence of sensorineural hearing loss in humans and animal studies (Meyerhoff et al., presented at Southern Section Triological Meeting, Jan. 1983) following use of these medications, otic drops remain the cornerstone of treatment for many infectious disorders of the ear. Twelve chinchillas underwent bilateral tympanostomy tube placement and daily instillation of Cortisporin Otic Suspension (polymyxin B, neomycin, hydrocortisone, propylene glycol) in the right external auditory canal for 7 consecutive days. The animals were sacrificed 3 days later, 10 days following initiation of the Cortisporin Otic Suspension treatment. Following routine preparation of the temporal bones for light microscopy, the tissue was evaluated for evidence of inflammatory changes. All 12 animals demonstrated granulation tissue, effusion and focal hemorrhage in the ears subjected to the Cortisporin Otic Suspension. All of the contralateral control ears were normal. The present data suggest that this inflammatory response is due to a topical irritant effect of the otic preparation.

Animals↗
PubMed1984

Visualization of acidic organelles in intact cells by electron microscopy.

We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

Antibodies, Monoclonal↗
PubMed1984

An otolaryngology surgical coding and reporting system.

A computerized information-gathering system has been developed at The University of Texas Health Science Center at Dallas for otolaryngologic head and neck surgery. Surgeons enter data on operations by answering multiple-choice questions at computer terminals situated near the operating rooms of each hospital. The system's main functions are to ensure completeness in the collection of operation data, allow easy review of such data for weekly conferences, and increase availability of such information for future comprehensive analysis. The scheme for data input, the logics of classifications, and the benefits of such a system are described.

Information Systems↗
PubMed1984

A surgical alternative to internal maxillary artery ligation for posterior epistaxis.

Posterior epistaxis is a disease of varying magnitude and is associated with considerable morbidity. Surgical management often consists of transantral ligation of the internal maxillary artery and its branches, with or without ligation of the ethmoidal arteries. Ten patients underwent an alternative surgical procedure in which the nasopharynx and posterior nasal cavity were indirectly examined for bleeding sites with a large laryngeal mirror. Nasal septal reconstruction with mobilization of the anterior cartilaginous septum from the maxillary crest allowed lateral displacement of the septum with excellent visualization of the lateral nasal walls. Hemorrhage was controlled by electrocoagulation of bleeding sites with a disposable, malleable suction electrocautery. No complications occurred in this group of 10 patients and no further epistaxis has been reported during a follow-up of 17 to 35 months.

Adolescent↗
PubMed1984

Non-auditory presentations of cerebellopontine angle lesions.

Most patients with cerebellopontine angle (CPA) neoplasms present with asymmetric hearing loss and/or tinnitus. For this reason it is well accepted that patients with these symptoms deserve a detailed neurotologic evaluation. Non-auditory presentations of CPA neoplasms, on the other hand, are frequently handled in a more complacent manner which may delay diagnosis and, therefore, result in increased morbidity. Six patients seen in the last 18 months serve to exemplify this point, as all 6 presented with non-auditory symptoms of CPA lesions and, in all cases, the diagnosis was delayed for at least 6 months. Any cranial neuropathy, unless otherwise explained, should suggest the presence of an intracranial or skull base lesion. The practicing physician must learn to give these non-auditory symptoms respect equal to that of asymmetric hearing loss and tinnitus and, therefore, equal evaluation.

Adult↗
PubMed1983

Mutant clone of Chinese hamster ovary cells lacking 3-hydroxy-3 -methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) converts HMG-CoA to mevalonate, a key intermediate in the formation of cholesterol and several nonsterol isoprenoid compounds. Using the bromodeoxyuridine/bisbenzimide photosensitization technique, we isolated a mutant clone of Chinese hamster ovary cells that requires mevalonate for growth. This mutant, designated UT-2 cells, expresses 2-5% of the HMG-CoA reductase activity of parental Chinese hamster ovary cells, even after growth for 9 months in the absence of selective pressure. By immunoblotting, no cross-reactive HMG-CoA reductase protein was detected in UT-2 cells. Incorporation of [14C]acetate and [14C]pyruvate into cholesterol was less than 5% of that in parental Chinese hamster ovary cells. In contrast, [3H]mevalonate incorporation into cholesterol was normal. The activities of acetoacetyl-CoA thiolase and HMG-CoA synthase, the two enzymes that precede HMG-CoA reductase in the cholesterol biosynthetic pathway, were normal or slightly elevated in UT-2 cells. No gross deletions or rearrangements in the gene for HMG-CoA reductase were apparent when DNA from UT-2 cells was digested with restriction endonucleases, subjected to Southern blotting, and probed with a 32P-labeled cDNA for HMG-CoA reductase. We conclude that UT-2 cells have a mutation that specifically prevents the production of normal amounts of HMG-CoA reductase.

Animals↗
PubMed1983

Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts.

Depletion of intracellular potassium (K+) caused a marked reduction in the rate of endocytosis of receptor-bound low density lipoprotein (LDL) and epidermal growth factor (EGF) in human fibroblasts. K+ could be depleted slowly by a 3-hr incubation of cells in isotonic K+-free buffer. Rapid K+ depletion was induced by incubation of cells for 5 min with hypotonic medium, followed by transfer to isotonic K+-free buffer. Within 30 min of this treatment, cellular K+ levels fell by more than 60%. When the K+ level fell below a threshold of 40% of normal, the number of coated pits declined by 80% and the rate of endocytosis of 125I-LDL decreased by 70 to 95% despite normal to increased receptor binding. Similar results were obtained with 125I-epidermal growth factor. Addition of KCl to the culture medium up to 2 hr after K+ depletion restored cellular K+ levels and returned endocytosis of 125I-LDL promptly to normal. RbCl was as effective as KCl, but CsCl, LiCl, and (CH3)4NCl had no effect. Restoration by KCl was blocked by ouabain, indicating that uptake via the Na+/K+ ATPase was required. These data demonstrate that depletion of intracellular K+ reversibly arrests coated pit formation and receptor-mediated endocytosis in human fibroblasts.

Cells, Cultured↗
PubMed1983

3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides.

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) is an abundant protein of the crystalloid endoplasmic reticulum of UT-1 cells, a line of cultured hamster cells that over-produces the reductase as a result of gene amplification. In the current studies, we show that reductase in UT-1 cells is a glycoprotein. The solubilized enzyme (Mr = 97,000) from UT-1 cells, Chinese hamster ovary cells, and rat liver was adsorbed quantitatively and specifically to concanavalin A-Sepharose. UT-1 cells incorporated [1,6-3H]glucosamine into the reductase; after release with endo-N-acetylglucosaminidase H most of the radioactivity was found in N-linked "high-mannose" chains, including Man6(GlcNAc)2, Man7(GlcNAc)2, and Man8(GlcNAc)2. The carbohydrate of the reductase was localized to a 30- to 35-kilodalton fragment that was separable proteolytically from a cytoplasmic 53-kilodalton fragment that contained the active site of the enzyme. We conclude that 3-hydroxy-3-methylglutaryl-CoA reductase is a transmembrane glycoprotein with an active site facing the cytoplasm and a carbohydrate-bearing site oriented toward the lumen of the endoplasmic reticulum.

Animals↗
PubMed1983

Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein.

Filipin has been widely used as an electron microscopic probe to detect 3-beta-hydroxysterols, principally cholesterol, in cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies have shown that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. This has led to the suggestion that these membranes are low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, we have carried out studies to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, we fixed the cells in either the presence or absence of filipin. This permitted us to identify all of the vesicles involved in the transport of LDL to the lysosome and to determine whether the membranes of these vesicles were able to bind filipin. We found that, coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. Furthermore, all of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. This suggests either that coated membrane contains normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.

Cholesterol↗
PubMed1983

Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells.

Mouse peritoneal macrophages can be induced to accumulate cholesteryl esters by incubating them in the presence of acetylated low density lipoprotein. The cholesteryl esters are sequestered in neutral lipid droplets that remain in the cell even when the acetylated low density lipoprotein is removed from the culture media. Previous biochemical studies have determined that the cholesterol component of cholesteryl ester droplets constantly turns over with a half time of 24 h by a cyclic process of de-esterification and re-esterification. We have used morphologic techniques to determine the spatial relationship of cholesteryl ester, free cholesterol, and lipase activity during normal turnover and when turnover is disrupted. Lipid droplets were surrounded by numerous 7.5-10.0-nm filaments; moreover, at focal sites on the margin of each droplet there were whorles of concentrically arranged membrane that penetrated the matrix. Histochemically detectable lipase activity was associated with these stacks of membrane. Using filipin as a light and electron microscopic probe for free cholesterol, we determined that a pool of free cholesterol was associated with each lipid droplet. Following incubation in the presence of the exogenous cholesterol acceptor, high density lipoprotein, the cholesteryl ester droplets disappeared and were replaced with lipid droplets of a different lipid composition. Inhibition of cholesterol esterification caused cholesteryl ester droplets to disappear and free cholesterol to accumulate in numerous myelin-like structures in the body of the cell.

Animals↗
PubMed1983

Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.

Adrenal Cortex↗
PubMed1983

Ultrastructural analysis of crystalloid endoplasmic reticulum in UT-1 cells and its disappearance in response to cholesterol.

The crystalloid endoplasmic reticulum (ER) consists of hexagonally packed membrane tubules that contain 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), an intrinsic membrane protein that catalyses the rate-limiting step in cholesterol synthesis. The crystalloid ER appears in a clone of Chinese hamster ovary cells, designated UT-1, that contain high levels of HMG CoA reductase as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. In the present studies, we have used ultrastructural morphometry to estimate that the crystalloid ER: (1) occupies about 15% of the volume of UT-1 cells; (2) contains 3.4-fold more membrane area than the plasma membrane; and (3) contains less than 700 subunits of HMG CoA reductase per micrometer2 of membrane surface. The crystalloid ER tubules contain 2000 intramembrane particles per micrometer2 with a mean diameter of 10.4 nm, as determined by freeze-fracture. The crystalloid ER membranes are low in cholesterol, as indicated by the small number of filipin-cholesterol complexes in freeze-fracture images after treatment with filipin. The addition of cholesterol or related sterols to UT-1 cells promoted a rapid and stepwise disappearance of the crystalloid ER. Initially, the crystalloid ER fragmented into randomly arranged vesicles and tubules. Subsequently, membrane-bound structures disappeared from the cell so that after incubation with cholesterol for 24-72 h, the cells appeared completely normal. We found no morphological evidence that autophagic vacuoles participate in the degradation. We conclude: (1) that the crystalloid ER is more extensive than necessary merely to support HMG CoA reductase; and (2) that upon exposure to cholesterol the crystalloid ER is degraded by a process that does not involve autophagy.

Animals↗
PubMed1983

Sudden sensorineural hearing loss.

Sudden sensorineural hearing loss is a frightening experience for the patient. The patient's worse fears concern tumors or stroke. Although the physician is often frustrated by a symptom whose cause at times is not apparent, every effort should be made to identify the causes of sudden sensorineural hearing loss and possible predisposing causes of idiopathic sudden sensorineural hearing loss. Therapy includes efforts to treat known causes either medically or surgically, to keep the patient at reduced physical activity, and depending upon the treating physician's own preference, attempts to favorably alter the ultimate prognosis through medical manipulation.

Hearing Loss, Sensorineural↗
PubMed1982

Receptor-mediated endocytosis and the cellular uptake of low density lipoprotein.

During receptor-mediated endocytosis various extracellular nutritional and regulatory molecules bind to plasma membrane receptors and rapidly enter target cells. In many systems (including those for certain plasma transport proteins, protein hormones, glycoproteins, toxins and viruses, and other plasma proteins) the receptors cluster in discrete regions of the surface membrane called coated pits, which invaginate into the cell to form endocytic vesicles. The extracellular ligand enclosed in the endocytic vesicle is delivered to intracellular sites, frequently to lysosomes, where it is degraded. In one system of receptor-mediated endocytosis, namely the one for plasma low density lipoprotein (LDL), the receptor functions to internalize LDL. The LDL is delivered to lysosomes where it is degraded and its cholesterol is released for use in the synthesis of membranes, steroid hormones and bile acids. Three recent advances in the LDL receptor system are reviewed: (1) the development of a method for purifying the receptor to apparent homogeneity and the demonstration that the LDL-binding site is contained within a glycoprotein of relative molecular mass 164000 and an acidic isoelectric point of 4.6; (2) the production of monoclonal antibodies directed against the receptor and the use of these antibodies as probes for receptor-mediated endocytosis; and (3) the use of monovalent carboxylic ionophores (such as monensin) to demonstrate by immunofluorescence that the LDL receptor enters the cell together with LDL, after which it recycles to the surface.

Animals↗