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Biomedical subjects

R G Johnson

Publications and source records attributed to R G Johnson.

At least 127 records · Page 7Linked to original sources
PubMed1991

Excitation-contraction uncoupling during ischemia in the blood perfused dog heart.

The bioluminescent of Ca(2+)-indicator, aequorin, was loaded into the left ventricular apex of blood-perfused hearts from 13 dogs for simultaneous recording of left ventricular pressure and intracellular calcium levels. During a 2 minute period of ischemia, systolic and diastolic pressures significantly decreased. In contrast, these pressure changes were associated with an increase in both systolic and diastolic calcium reaching a maximum diastolic value of 0.59 microM and a systolic value of 1.11 microM. This apparent dissociation between pressure and [Ca2+]i supports the hypothesis that changes in myofilament Ca2+ responsiveness are of major importance in modulating contractility during ischemia in large mammalian hearts.

Aequorin↗
PubMed1991

Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis.

Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.

Adenosine Triphosphate↗
PubMed1991

Early ostial vein graft stenosis: management by atherectomy.

During the initial perioperative period (1 mo to 1 yr) after saphenous vein coronary grafting, early stenosis and occlusion occurs in 5-8% of grafts due to intimal hyperplasia. We report a patient who developed ostial stenosis within 4 mo of bypass surgery at the aortotomy site of two vein grafts. Balloon angioplasty of the elastic stenoses did not provide significant luminal enlargement, but successful treatment of the lesions was obtained using directional atherectomy. Histological examination demonstrated intimal hyperplasia. Directional atherectomy may be an excellent technique for treatment of elastic ostial vein graft stenoses in lieu of conventional balloon dilatation.

Aged↗
PubMed1991

Sodium flux and bioenergetics in the ischemic rat liver.

Concurrent 23Na and 31P nuclear magnetic resonance spectroscopy has been employed to study the effects of ischemia upon the high-energy phosphagens and sodium ion concentration within the in vivo rat liver. High-energy phosphates in the form of ATP were depleted within 10 min of the onset of ischemia when measured by NMR. However, similar liver samples subjected to analytical biochemistry retained 27 +/- 12% of their ATP after a similar 10-min ischemic insult. Time-dependent 23Na NMR measurements, obtained in the presence of the shift reagent Dy(TTHA) to distinguish intracellular from extracellular sodium, revealed a rapid rise in the intracellular sodium when the liver was made ischemic. Intracellular and extracellular sodium concentrations approached equilibrium with an exponential time constant of 14.7 +/- 7 min. The initial rate of sodium influx was calculated to be 1.50 meq.l-1.min-1. The results indicate that the ischemic liver has a high passive sodium permeability and that NMR detectable 31P signals reflect the actual availability of cytosolic high-energy phosphates to enzymes, in this instance the membrane-bound [Na+, K+]-ATPase.

Adenosine Triphosphate↗
PubMed1991

Enhanced gap junction formation with LDL and apolipoprotein B.

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

Animals↗
PubMed1991

MP26, a protein of intercellular junctions in the bovine lens: electrophoretic and chromatographic characterization.

We have characterized the membrane protein of apparent molecular weight 26 kD from bovine lenses (MP26 or MIP) with respect to six different electrophoretic and chromatographic procedures. These include one- and two-dimensional gel electrophoretic procedures, as well as SDS-hydroxylapatite chromatography. The two-dimensional gels include isoelectric focusing with both conventional ampholytes and buffer focusing methods. With buffer focusing, the membranes are solubilized without the use of SDS and the isoelectric focusing is performed in the absence of SDS. As specific probes for MP26, a monoclonal antibody and an anti-MP26 rabbit serum were used, the latter prepared against electrophoretically purified MP26. These separation techniques were adapted to MP26 in order to permit a more detailed characterization of this protein and to search for any heterogeneity in this size range, specifically other junctional proteins or protein fragments. We have found evidence for charge heterogeneity in MP26, but no evidence for multiple membrane proteins of Mr 26,000 in urea-treated membranes. The charge heterogeneity appears to be related to a phosphorylation of MP26. The results reported here aid the interpretation of a variety of data, especially findings on the reconstitution of MP26 in artificial membranes and results from work with polyclonal MP26 antibodies. These investigations are all designed to evaluate the proposed role of MP26 as a protein of cell-to-cell channels in the lens fiber cell.

Animals↗
PubMed1991

IgM-antibody response to hepatitis C virus antigens in acute and chronic post-transfusion non-A, non-B hepatitis.

A specific IgM solid-phase enzyme-linked immunoassay for the diagnosis of a recent infection by hepatitis C virus (HCV) was developed. The assay utilizes a structural antigen encoded by sequences at the 5' end of HCV (core region) and non-structural (NS) antigens encoded by the NS-3 (33c) and NS-4 (c100-3) regions of the HCV genome. Serial serum samples from several clinically diagnosed post-transfusion non-A, non-B hepatitis patients were analyzed for anti-HCV IgM. This antibody was frequently but transiently detected. Anti-HCV core IgM was more frequently detected than anti-c100-3 or anti-33c IgM. In individuals who resolved their HCV infection or progressed to chronicity, anti-HCV IgM was produced transiently at or near the onset of clinically diagnosed acute hepatitis.

Acute Disease↗
PubMed1991

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.

Animals↗
PubMed1991

Detection of antibodies to hepatitis C virus in U.S. blood donors.

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.

Amino Acid Sequence↗
PubMed1991

Regulation of the secretory response in bovine chromaffin cells.

The nicotine-induced current and the Ca2+ current were studied in cultured bovine chromaffin cells using the whole cell patch-clamp technique. The dose-response curve for the nicotinic current gave a dissociation constant of 53 microM and a Hill coefficient of 1.3. Desensitization of the nicotinic current was rapid, with time constants of 22 and 155 ms at 10 microM nicotine. At higher concentrations of nicotine, both time constants decreased somewhat, but the most prominent effect was on the ratio of the two components. Recovery from desensitization was fitted by a single exponential with a time constant of approximately 6 s. Ca2+ current and catecholamine secretion were highly sensitive to changes in extracellular H+ concentration ([H+]o), such that small increases in [H+]o markedly decreased both. The Ca2+ current measured in a chromaffin cell located within a cluster of cells, but not in a single isolated cell, was markedly suppressed when KCl or nicotine was used to induce secretion, suggesting possible local feedback of secretory agents. Among agents secreted by chromaffin cells, ATP, enkephalins, epinephrine, and protons, only protons significantly suppressed the Ca2+ current. Our findings suggest that the secretory response of chromaffin cells may be modulated by rapid desensitization of the nicotinic receptor and a secretion-dependent suppression of the Ca2+ current.

Adrenal Medulla↗
PubMed1991

Effects of vasoactive drugs on flows through left internal mammary artery and saphenous vein grafts in man.

Vasoactive agents are commonly used in the postcardiopulmonary bypass period to elevate the mean arterial pressure of myocardial revascularization patients. Concern exists that administration of vasoactive agents in this setting may affect flow through saphenous vein and internal mammary artery grafts. Twenty-eight patients were randomly assigned to receive one of the six two-drug combinations of phenylephrine, norepinephrine, and epinephrine. After termination of cardiopulmonary bypass baseline, hemodynamic measurements and electromagnetic flow probe measurements of saphenous vein and internal mammary artery graft flow were made. The first agent was then infused to elevate mean arterial pressure 20 mm Hg. After 5 minutes of stability, hemodynamic and graft flow measurements were repeated. The infusion was terminated, 5 minutes of stability were obtained, and baseline measurements were repeated. The second agent was then infused, and measurements were repeated after a 5-minute stabilization period. Phenylephrine induced a nonsignificant increase in saphenous vein graft flow (68 +/- 31 versus 81 +/- 49 ml/min) and a significant decrease in internal mammary artery graft flow (40 +/- 16 versus 32 +/- 12 ml/min). Norepinephrine induced a significant increase in saphenous vein graft flow (80 +/- 39 versus 97 +/- 39 ml/min) and no significant change in internal mammary artery graft flow (44 +/- 20 versus 45 +/- 20 ml/min). Epinephrine induced a significant increase in both saphenous vein (82 +/- 38 versus 96 +/- 40 ml/min) and internal mammary artery (38 +/- 12 versus 55 +/- 24 ml/min) graft flows. We conclude that administration of vasoactive agents in the postcardiopulmonary bypass period may significantly affect saphenous vein and internal mammary artery graft flows.

Adrenergic alpha-Agonists↗
PubMed1990

Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes.

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.

Amino Acid Sequence↗
PubMed1990

Lipids in gap junction assembly and function.

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions. The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

Animals↗
PubMed1990

Aortic valve operation after percutaneous aortic balloon valvuloplasty.

From January 1986 to May 1988, 179 patients underwent percutaneous aortic balloon valvuloplasty at our institution. Forty-five (25%) patients subsequently required operation at this institution or other institutions. The indication for percutaneous aortic balloon valvuloplasty was patient preference in 15 and an estimation of excessive operative mortality in 30. Three patients required emergency operation immediately after valvuloplasty, and the other 42 had an elective operation a mean of 7.8 months afterward. All but 3 of these patients had major progression of symptoms, and all had valvular restenosis. In spite of the high-risk status of these patients, there were only four hospital deaths among the 45 patients. Three additional patients have died a mean of 11.4 months postoperatively. The condition of all survivors remains clinically improved after the valve operations. Percutaneous aortic balloon valvuloplasty must be recognized as a palliative procedure, and should be reserved for patients who are truly not candidates for operation or situations in which a brief period of reduction in gradient might improve a patient's condition before operation.

Aged↗
PubMed1990

The early response of articular cartilage to ACL transection in a canine model.

Ten outbred dogs underwent unilateral transection of the anterior cruciate ligament of the knee (stifle) joint with sacrifice from 6 to 27 weeks post-operatively. On the operated side were found mild degenerative changes histologically and evidence of increased proteoglycan synthesis with no net loss of uronic acid. Water content was increased in the medial femoral condyle. Contralateral knees showed evidence of increased proteoglycan synthesis and uronic acid content without histologic cartilage lesions.

Animals↗
PubMed1990

Acetylcholine and ATP are coreleased from the electromotor nerve terminals of Narcine brasiliensis by an exocytotic mechanism.

Although the exocytotic mechanism for quantal acetylcholine (ACh) release has been widely accepted for many years, it has repeatedly been challenged by reports that ACh released upon stimulation originates from the cytosol rather than synaptic vesicles. In this report, two independent experimental approaches were taken to establish the source of ACh released from the electromotor system of Narcine brasiliensis. Since ATP is colocalized with ACh in the cholinergic vesicle, the exocytotic theory predicts the corelease of these two components with a stoichiometry identical to that of the vesicle contents. The stimulated release of ATP from isolated synaptosomes could be accurately quantitated in the presence of the ATPase inhibitor adenosine 5'-[alpha, beta-methylene]triphosphate (500 microM), which prevented degradation of the released ATP. Various concentrations of elevated extracellular potassium (25-75 mM), veratridine (100 microM), and the calcium ionophore ionomycin (5 microM) all induced the corelease of ACh and ATP in a constant molar ratio of 5-6:1 (ACh/ATP), a stoichiometry consistent with that established for the vesicle content. In parallel to these stoichiometry studies, the compound 2-(4-phenylpiperidino)cyclohexanol (AH5183) was used to inhibit specifically the vesicular accumulation of newly synthesized (radiolabeled) ACh without affecting cytosolic levels of newly synthesized ACh in cholinergic nerve terminals. Treatment with AH5183 (10 microM) was shown to inhibit the release of newly synthesized ACh without markedly affecting total ACh release; thus, the entry of newly synthesized ACh into the synaptic vesicle is essential for its release. We conclude that ACh released upon stimulation originates exclusively from the vesicular pool and is coreleased stoichiometrically with other soluble vesicle contents.

Acetylcholine↗
PubMed1990

Activation of the Na+, K(+)-ATPase in Narcine brasiliensis.

The in vivo activation and turnover rates of the sodium pump (Na+, K(+)-ATPase) were investigated in the electrocytes of the electric organ of the elasmobranch Narcine brasiliensis. The Narcine electric organ appears to be an excellent model for the study of sodium pump activation in an excitable tissue. The sodium transmembrane gradient and high-energy phosphagens were concurrently measured by 23Na and 31P NMR spectroscopy. The resting electric organ, which depends primarily on anaerobic metabolism, displays a high concentration of phosphocreatine (PCr). It has an intracellular sodium concentration ([Na+]i) of 20 +/- 10 milliequivalents/liter as estimated by NMR. Electrical stimulation of the nerves innervating the electric organ results in an increase in [Na+]i in the electrolyte and rapid depletion of PCr. Ouabain causes an 85% decrease in utilization of high-energy phosphagens, indicating that rapid PCr turnover in this tissue is mainly due to Na+, K(+)-ATPase activity. From these data we can determine that the rate of sodium pump turnover increases by greater than 3 orders of magnitude within several hundred milliseconds. In excised unstimulated electric organ slices, changes in [Na+]i equivalent to those occurring with stimulation, but induced by hyperosmolar conditions, do not result in increased PCr hydrolysis. We conclude that cholinergic stimulation of the electric organ causes a rapid and extremely large increase in sodium pump turnover, which is regulated predominantly by factors other than [Na+]i.

Animals↗