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B R Copeland

Publications and source records attributed to B R Copeland.

27 records · Page 2Linked to original sources
PubMed1984

Role of membrane potential in protein folding and domain formation during secretion in Escherichia coli.

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.

Bacterial Proteins↗
PubMed1982

Renaturation and identification of periplasmic proteins in two-dimensional gels of Escherichia coli.

The locations of the periplasmic proteins of Escherichia coli on standard two-dimensional gel patterns are described. The periplasmic fractions were prepared by osmotic shock of plasmolyzed whole cells and by release during EDTA-lysozyme treatment of whole cells. Within this fraction of proteins, we identify nine binding proteins (leucine-specific, glutamate-aspartate, glutamine, cystine, galactose, maltose, xylose, ribose, and arabinose) in addition to leucine-isoleucine-valine binding protein, which has been previously identified (Bloch, P. L., Phillips, T. A., and Neidhardt, F. C. (1980) J. Bacteriol. 141, 1409-1420). The identifications are based upon genetic criteria, protein induction, and comigration with purified protein. The levels of these proteins are compared in strains K12, B, and HA12 (a derivative of W). A technique was developed for renaturation of the ligand binding sites of periplasmic binding proteins in denaturing two-dimensional gels. This technique was used to demonstrate that leucine-specific and cystine binding proteins both have different isoelectric points in different strains. Renaturation was also used to demonstrate that there are two different charged forms for glutamine binding protein.

Carrier Proteins↗
PubMed1982

A theory of effect of protons and divalent cations on phase equilibria in charged bilayer membranes: comparison with experiment.

We summarize the concepts in the recently developed statistical mechanical theory of the effects of proton binding and divalent cation binding on phase equilibria in bilayer membrane composed of acidic phospholipids. The theory is used to calculate membrane phase transition temperatures for different aqueous concentrations of protons, divalent cations and monovalent salt. We discuss methods for calculating transition temperatures even for systems in which there is not an excess of protons or divalent cations relative to lipids. The results are compared with existing experimental data for a number of lipids. There is good agreement between calculated transition temperature vs. pH curves and experimental data for dimyristoylmethylphosphatidic acid, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylserine, and dimyristoylphosphatidic acid. General thermodynamic considerations are used to derive in Clapeyron-like equation for the rate of variation in membrane transition temperature with divalent cation concentration. This equation and some available experimental data are used to argue that the large increase in solid to fluid phase transition temperature that is observed experimentally as the divalent cation concentration is increased is the result of the metastable solid phase that exists at low but not high divalent cation concentration. A calculated coexistence diagram is compared with existing experimental data for transition temperatures of dimyristoylphosphatidylglycerol membranes at different total calcium concentrations. Good agreement is obtained when the existence of a metastable solid phase is assumed.

Cations, Divalent↗
PubMed1982

High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.

Acantholysis↗
PubMed1980

Freeze-fracture of reconstituted model membranes used as targets for cell-mediated cytotoxicity.

In a recent communication (Hollander, N., Mehdi, S.Q., Weissmann, I.L., McConnell, H.M. and Kriss, J.P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4042-4045) we reported that reconstituted model membranes containing murine tumor cell membrane proteins can be substituted for living cells as targets for cell-mediated cytolysis by allosensitized T-lymphocytes. The specificity of the lytic process was governed by the appropriate histocompatibility antigen (H-2). It was stressed, however, that although a standard protocol was faithfully followed for the reconstitution of the target membrane vesicle, the system was not uniformly reproducible. Some experiments showed high levels of specific vesicle killing while no lysis was observed in others. This work extends our description of the structural requirements of reconstituted membrane vesicles.

Animals↗
PubMed1980

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol.

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0--20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.

Cholesterol↗
PubMedDate not supplied

The beneficial effect of intracarotid urokinase on acute stroke in a baboon model.

The capacity of intracarotid infusion of urokinase to salvage neurologic function in a baboon model of acute thrombotic stroke has been studied. The model consists of reversible eccentric balloon compression (3 hours) of the right middle cerebral artery (MCA) proximal to the take-off of the lenticulostriate arteries (LSA), resulting in in situ thrombosis of perforating branches supplying the right corpus striatum. Neurologic endpoints included quantitative assessment of neurologic function (NE), estimation of cerebral infarction volume by computerized tomographic (CT) scan, and carotid angiography. In untreated acute stroke control animals (n = 6), a persistent decrease in functional score (from 100 to 36 +/- 11) at 14 days and a defined region of cerebral infarction (volume = 3.2 +/- 1.5) were detected at 10 days. Intracarotid urokinase administered to five animals (1.2 X 10(6) IU over 60 min) following the 3 hour period of MCA occlusion improved neurologic function (NE = 50, 55, 85, 100, 100) and reduced infarction size (0.3, 0.5, 0.8, 0.7, 1.1 cm3, respectively) without evidence of intracranial hemorrhage. Systemic fibrinogenolysis was produced in all five treated animals. We conclude that thrombolytic therapy given within 3 hours of experimental thrombotic occlusion may salvage neurologic function and reduce cerebral infarction volume without CT scan detectable intracranial bleeding.

Acute Disease↗
PubMedDate not supplied

Experimental acute thrombotic stroke in baboons.

To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9 cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8. Similar 111In-platelet imaging and histologic scoring experiments carried out in four animals pretreated with the antithrombotic combination heparin and ticlopidine showed marked reduction of both 111In-platelet activity (0.01 +/- 0.03 ml vs. 0.55 +/- 0.49 ml; p less than 0.01) and thrombotic occlusion of the microcirculation (10.8 +/- 7.4 units vs. 34.8 +/- 2.8 units; p less than 0.01) in the right corpus striatum following 3 hours of MCA occlusion. In separate control experiments 111In-labeled autologous platelets were infused after the 3 hour period of right MCA occlusion and subsequent balloon deflation in two animals; no focus of 111In-platelet activity was demonstrated in fixed whole brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗