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Biomedical subjects

R G Johnson

Publications and source records attributed to R G Johnson.

At least 19 recordsLinked to original sources
PubMed1992

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.

Animals↗
PubMed1992

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.

Animals↗
PubMed1992

War in Croatia.

Explore the source record for details and available documents.

Croatia↗
PubMed1992

Endothelial modulation of porcine coronary microcirculation perfused via immature collaterals.

Porcine hearts have relatively few native collateral vessels and lack the propensity to develop normal perfusion to the collateral-dependent myocardium. To examine microvascular responses in the collateral-dependent region, collateral vessels were stimulated in pigs by the Ameroid constrictor technique. After 4-7 wk, isolated microarterial vessels (90-170 microns ID) were studied in a pressurized (40 mmHg), no-flow state. Microvessels from noninstrumented pigs were used as controls for vascular studies. Although myocardium in the collateral-dependent region showed minimal evidence of infarction, percent systolic shortening was reduced at rest and after pacing compared with myocardium in the normally perfused region. Relaxations to the receptor-mediated endothelium-dependent agents ADP and bradykinin were impaired in collateral-dependent coronary microvessels. Relaxations to the calcium ionophore A23187, which acts through a non-receptor-mediated mechanism, were similar in control and Ameroid microvessels. Relaxations to the endothelium-independent agent sodium nitroprusside were markedly enhanced in microvessels from the collateral-dependent region compared with microvessels from control hearts. In conclusion, receptor-mediated endothelium-dependent relaxation is impaired and endothelium-independent relaxation to sodium nitroprusside is enhanced in microvessels from myocardium perfused by immature collateral vessels.

Animals↗
PubMed1992

Comparison of two transfusion strategies after elective operations for myocardial revascularization.

We performed a prospective, randomized trial of two different strategies for postoperative packed red blood cell replacement in 39 autologous blood donors undergoing elective myocardial revascularization. The "liberal" group received blood to achieve a hematocrit value of 32%, and the "conservative" group received transfusions for a hematocrit value less than 25%. Although the groups had significantly different mean hematocrit values from the fourth postoperative hour (28.7% versus 31.2%) through the fifth postoperative day (28.4% versus 31.3%), there were no significant differences in fluid requirement, hemodynamic parameters, or hospital complications. Significantly fewer units of packed cells were required in the conservatively transfused group (20 units/20 patients) compared with the liberally transfused group (37 units/18 patients) (p = 0.012). Exercise tests were performed on the fifth and sixth postoperative days, with a transfusion being given to the conservative group between tests. Although a significant improvement in exercise endurance occurred in the conservative group receiving a transfusion (p = 0.008), no significant difference in duration or degree of exercise was demonstrated between the two groups on either day. In comparing these two groups of profoundly anemic patients, we identified no adverse consequence associated with the greater degree of hemodilution and could identify no correlation between hematocrit value and exercise capacity. We conclude that although the limits of hemodilution are still poorly defined, postoperative blood transfusion in revascularized patients should be guided by clinical indications and not by specific hematocrit values.

Anemia↗
PubMed1992

Mechanical assistance of the left ventricle: acute effect on cardiac performance and coronary flow of different perfusion patterns.

Mechanical circulatory assistance by ventricular assist devices provides an opportunity to influence the aortic pressure pattern, which may affect ventricular loading and coronary perfusion. The effect of synchronous, pulsatile coronary perfusion of an assist device-supported left ventricle has not been studied. To analyze the effect of different perfusion patterns on left ventricular performance and on coronary flow, independent of pressure and volume loading, we used three different modes of aortic perfusion in an isometric, contracting, isolated canine heart model. The effect of nonpulsatile, counter-pulsatile, and copulsatile coronary perfusion was analyzed in four subgroups to simulate different, clinically relevant situations (using two different ventricular end-diastolic volumes [normal and high] and two mean perfusion pressures [normal and critically low]). Our experiments demonstrated that total coronary flow is optimized by making the perfusion pressure pulsatile and by synchronously timing the pump systole with ventricular diastole (counterpulsation). Under identical conditions of preload and mean perfusion pressure, coronary flow and left ventricular contractility were decreased during non-pulsatile and copulsatile aortic perfusion when compared with counterpulsatile flow. There were no significant differences between the nonpulsatile and copulsatile modes. We conclude from these data that a nonejecting, but contracting, left ventricle will have improved systolic function and coronary blood flow if the coronary perfusion pressure is synchronized in a counterpulsatile manner. This is a significant implication for mechanical left ventricular assist devices when used to promote myocardial recovery.

Animals↗
PubMed1992

Pnemostasis of injured lung in rabbits with gelatin-resorcinol formaldehyde-glutaraldehyde tissue adhesive.

Peripheral bronchopulmonary fistulas, or parenchymal air leaks, may occasionally be resistant to conventional suture or stapling techniques. In such instances, the tissue is poor and the associated morbidity of a persistent leak will be the greatest. We tested a previously described tissue adhesive compounded from gelatin resorcinol and polymerized at the time of application with a glutaraldehyde-formaldehyde mixture. We evaluated the ability of the adhesive to seal incisional air leaks acutely in the lungs of rabbits under conditions of positive intratracheal pressure and persistent ventilation. We also tested the efficacy of the glue at intervals after compounding to assess the shelf life of this nonproprietary formula. We established a technique for application of this adhesive and demonstrated its ability to decrease consistently the magnitude of air leaks, while generally providing complete pneumostasis in the presence of clinically relevant positive pressure ventilation. We conclude that this material can be prepared months in advance of its occasional use as an effective means of dealing with parenchymal air leaks encountered at operation.

Animals↗
PubMed1991

Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays.

BACKGROUND: The causes of post-transfusion non-A, non-B hepatitis are still not fully defined, nor is it clear how accurate the tests are that are used to screen blood donors for hepatitis C virus (HCV) and to diagnose post-transfusion hepatitis caused by infected blood. METHODS: We used two first-generation enzyme-linked immunoassays (EIAs) and one second-generation immunoassay to test for anti-HCV antibodies in serum samples collected between 1976 and 1979 in the Transfusion-Transmitted Viruses Study (from 1247 patients who underwent transfusion and 1235 matched control subjects who did not receive transfusions). We tested serum collected before and after infection from the patients in whom non-A, non-B hepatitis developed, serum from their blood donors, and serum from 41 of the control subjects who had hepatitis unrelated to transfusion. RESULTS: Of the 115 patients in whom post-transfusion non-A, non-B hepatitis developed, the initial serum samples of 111 were anti-HCV-negative; after hepatitis developed in these 111 patients, the first-generation EIAs detected anti-HCV in 51 (46 percent), and the second-generation assay detected anti-HCV in an additional 16 (14 percent), for a total of 60 percent. Of 40 controls, 37 were anti-HCV-negative initially, and none seroconverted after hepatitis developed. If the 3 percent rate of non-A, non-B, non-C hepatitis among the controls (37 of 1235) was applied to the 1247 transfusion recipients, only 74 of the 111 cases of hepatitis were attributable to the transfusion. Thus, 91 percent (67 of 74) of the cases of post-transfusion hepatitis were caused by HCV. Of the 99 donors, 60 were HCV-positive (9 on second-generation tests only) and 39 were not. CONCLUSIONS: Nearly all cases of non-A, non-B post-transfusion hepatitis are caused by HCV. Screening with a second-generation assay improves the rate of detection of HCV infection in patients with post-transfusion hepatitis and in blood donors. The use of this test showed a 3.6 percent risk of non-A, non-B, non-C hepatitis, which was not significantly different from the rate in the controls (3.0 percent).

Blood Donors↗
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↗