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Biomedical subjects

R G Johnson

Publications and source records attributed to R G Johnson.

At least 163 records · Page 9Linked to original sources
PubMed1987

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression.

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.

Animals↗
PubMed1987

Two-dimensional and Doppler echocardiographic diagnosis of an aortic to right atrial fistula complicating aortic dissection.

A 60 year old woman presented with massive aortic root dilation and sudden cardiovascular collapse 10 years after aortic valve replacement. An aortic to right atrial fistula was diagnosed by echocardiographic imaging and Doppler ultrasound. At operation, the patient was found to have chronic aortic dissection with aneurysm formation. Rupture of the aneurysm into the right atrium was confirmed.

Aortic Dissection↗
PubMed1987

Dallas discogram description. A new classification of CT/discography in low-back disorders.

A new classification method for CT/discography was developed. The Dallas discogram description (DDD) related five separate categories of information. Degeneration and annular disruption were regarded as separate phenomena. Additionally, provoked pain response, contrast volume, and miscellaneous information were recorded. Discogram findings of 59 patients with low-back and/or leg pain were graded according to the new method and compared with standard methods using routine anteroposterior and lateral discographic images. Findings from routine discography and CT/discography were graded and correlated with myelographic and plain computerized axial tomography (CAT) scans. This study demonstrated that the contrast-enhanced axial view provided by CT/discography served as a useful projection for demonstrating disc pathology. CT/discography analyzed according to DDD offered a more sensitive discriminator of disc degeneration from annular disruption (disc protrusion/leaking). This evaluation can be recommended as the procedure of choice when revision of spine surgery is considered or when there is an equivocal or negative correlation between clinical information and myelography or CAT scan.

Adult↗
PubMed1987

The relationship of pain provocation to lumbar disc deterioration as seen by CT/discography.

The CT/discographic findings from 225 discs in 91 low-back pain patients were compared to the pain provocation during the injection of contrast into the disc. The radiographic appearance of disc deterioration demonstrating disc degeneration and annular disruption of each disc was classified separately using a fourpoint scale: normal, slight, moderate, or severe. Pain reaction to the discogram at each level was recorded as follows: no pain, dissimilar pain, similar pain, or exact reproduction of the patient's clinical pain. This more precise analysis demonstrated a significant relationship between pain and deterioration of discs. The CT/discogram presents an axial view of the disc that allows a subgrouping of disc deterioration that can discriminate between peripheral deterioration (degeneration) and internal deterioration (disruption). The disruption supposedly occurs earlier and is more likely to be the source of exact pain reproduction.

Adult↗
PubMed1987

Proton pumps and chemiosmotic coupling as a generalized mechanism for neurotransmitter and hormone transport.

Neuroendocrine secretory vesicles contain within their membranes a highly specialized H1-translocating ATPase responsible for the generation and maintenance of an electrochemical proton gradient, delta pH inside acidic, and delta psi inside positive. Coupled with a high internal buffering capacity and extremely low permeability of the membrane to protons, this proton pump can generate and maintain an intravesicular pH of 5.5, independent of the external pH, and transmembrane electrical potential of 60 mV. The chemiosmotic gradient has important implications for several functions of the secretory vesicles: (1) maintaining oxidizable substances (such as biogenic amines) in the unoxidized form; (2) stimulating (or inhibiting) peptide processing enzymes; (3) permitting precipitation of intravesicular protein complexes, thereby increasing the amount that can be stored within the vesicle; and (4) serving as the driving force for the uptake of certain hormones and neurotransmitters such as acetylcholine, biogenic amines, and ATP. By using the putative biogenic amine transporter as an example, it can be demonstrated that based purely upon the existence of a transporter in equilibrium with the electrochemical proton gradients, an amine concentration approaching 135,000 to 1 can be achieved. The bioenergetics of amine transport do not predict the molecular mechanism of amine translocation. By using kinetic analyses of amine accumulation under a variety of situations, however, initial information concerning the salient aspects of amine transport is being obtained.

Animals↗
PubMed1986

Lung lamellar bodies maintain an acidic internal pH.

The internal pH of lung lamellar bodies was investigated with membrane permeable basic amines. Isolated granular pneumocytes and isolated lung lamellar bodies exhibited fluorescence when exposed to 8 microM quinacrine, suggesting accumulation of this dye due to an acidic internal pH. Uptake of [14C]methylamine by isolated lung lamellar bodies was measured to quantitate the intralamellar body pH. In KCl-ATP (10 mM) medium (pH 7.0), the accumulation ratio of methylamine (inside/outside) during 2-min incubation was 8.1 +/- 0.47 (mean +/- S.E., n = 8) indicating an internal pH of 6.1. Lamellar bodies accumulated methylamine almost 30-fold in K+-free mannitol medium indicating an internal pH of 5.6. The pH gradient across the lamellar body membrane decreased when external pH was decreased or when ATP was omitted. The pH gradient was also decreased by the addition of 10 mM NH4Cl, 2 micrograms/ml nigericin, 0.02 mM N,N'-dicyclohexylcarbodiimide, or 1 mM N'-ethylmaleimide. These observations indicate that lamellar bodies maintain an acidic interior (pH 6.1 or below) which is generated by an energy-dependent process.

Animals↗
PubMed1986

Phosphorylation of lens intrinsic membrane proteins by protein kinase C.

Two intrinsic proteins of bovine lens membranes with apparent relative molecular masses (Mr, app) of 26,000 and 18,000 were phosphorylated in intact membranes by protein kinase C prepared from either bovine brain or lens. The kinase preparations exhibited histone H1 phosphorylation dependent on calcium and phospholipid but not on cAMP. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the lens membranes showed a major band at Mr, app = 26,000 (identified as MP26, the main intrinsic protein of lens fiber cells), an intermediate band at Mr, app = 18,000 and several minor bands. Autoradiography of complete assay mixture containing protein kinase C, calcium, magnesium and [gamma-32P]ATP showed major bands at Mr, app = 18,000 and 26,000. Several lines of evidence indicated that the label at Mr, app = 26,000 was associated with MP26, a protein which has been found in lens junctions and which may form cell-cell channels. Treatment of the phosphorylated membranes with chymotrypsin and V8 protease cleaved the major band at Mr, app = 26,000 to fragments of Mr, app .= 22,000 and 24,000. Label was not detected in the resulting Mr, app = 22,000 peptide, but the Mr, app = 24,000 peptide was found to be labeled. Phosphoamino acid analysis of MP26 indicated that approximately 75% of the label was on phosphoserine and 25% was on phosphothreonine. No label was found on phosphotyrosine. These results differ from those reported for cAMP-dependent phosphorylation of lens proteins. Phosphorylation by protein kinase C may account for some of the labeling of MP26 detected in vivo.

Amino Acids↗
PubMed1986

Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes.

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.

Animals↗
PubMed1986

Successful management of aortic valve endocarditis with associated periannular abscess and aneurysm.

Aortic valve endocarditis extending beyond the aortic root presents a particularly difficult management problem. During a four-year period we have seen four patients with extensive annular abscesses involving the native valve (in 1 patient) and prosthetic valves (in 3). In all these patients the aortic roots were debrided and valved conduits were replaced with coronary artery grafts while appropriate antibiotics were administered. Postoperative complications included conduction abnormalities, hemorrhage, and low cardiac output, but all 4 patients were discharged from the hospital. Two patients died, 1 of an unrelated disease at 13 months postoperatively and 1 of endocarditis at 16 months postoperatively. The two remaining patients are alive and well at 11 and 46 months postoperatively. We conclude that, in spite of the prosthetic material required, this procedure permits adequate debridement and safe reconstruction for the rare patient in whom endocarditis has resulted in aortic-ventricular discontinuity.

Abscess↗
PubMed1986

Transection of the canine anterior cruciate ligament: a concise review of experience with this model of degenerative joint disease.

Transection of the anterior cruciate ligament in dogs is probably the most widely used model for degenerative articular disease. Two techniques, the percutaneous stab and arthrotomy, are currently employed. Reported results of the degree of cartilage damage and the time of onset of lesions vary considerably in the literature. Several papers described overt fibrillation as early as 6 weeks post-operatively while others found only mild changes up to nearly 4 years. Critical analysis of the model revealed that parameters such as the breed of dog, its age, weight, amount of exercise and technique of surgery may influence the onset and severity of lesions. Further research is essential to define the relative importance of each.

Age Factors↗
PubMed1986

A lens intercellular junction protein, MP26, is a phosphoprotein.

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.

Amino Acids↗
PubMed1986

Effect of preload on ischaemic and non-ischaemic left ventricular regional function.

The response to preload of ischaemic and non-ischaemic regions of the left ventricle was studied in 14 dogs undergoing right heart bypass with mean aortic pressure and heart rate held constant. Regional function was measured by sonomicrometry before and after coronary artery occlusion. In the ischaemic region, as expected, there was paradoxical systolic lengthening (that is, systolic shortening was negative) but as stroke volume was progressively increased end diastolic length increased, whereas end systolic length changed little; thus systolic lengthening decreased (systolic shortening increased). Ischaemic regions that were dyskinetic at low stroke volumes were virtually akinetic at high stroke volumes. Additional studies showed that this response was not attributable to increased regional blood flow at high preloads and occurred over a wide range of heart rates and mean aortic pressures. Plots of systolic shortening against end diastolic length, expressing the regional Frank-Starling relation, were well described by linear regression in both ischaemic and non-ischaemic regions, although a few of these relations were better described by higher order polynomials. The slopes of these relations in the ischaemic region were 0.86(0.05) before and 0.83(0.06) after ligation, reflecting a small effect of preload on end systolic length. The data suggest that when contractility and afterload are constant preload determines the magnitude and in certain instances the sign of systolic shortening. In any ischaemic regions incapable of developing force the positive slope of the Frank-Starling relation is attributable to myocardial passive elastic properties. Paradoxical lengthening does not, however, necessarily indicate the absence of active force development; positive and negative values of systolic shortening describe a continuous spectrum of regional contractility. Thus the effects of preload and contractility on systolic shortening in ischaemic as well as non-ischaemic myocardium require differentiation.

Animals↗
PubMed1986

Effects of isobutyl-2-cyanoacrylate on bone healing.

An in vivo evaluation of isobutyl-2-cyanoacrylate as an osseous adhesive was performed to determine its effect on bone healing. Oblique cortical fractures were induced at the tibial-fibular junctions of adult rabbits. Forty-eight fractures were repaired by the use of isobutyl-2-cyanoacrylate and a full cerclage wire. The opposite control fracture was repaired with a full cerclage wire only. One hundred percent of the glued fractures were not united through the glue interface, although normal periosteal new bone formation (secondary bone healing) took place around the glue. One hundred percent of the nonglued fractures healed by secondary bone union. Reactions were not seen in the bone or soft tissue adjacent to the adhesive material.

Animals↗
PubMed1985

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens.

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.

Affinity Labels↗
PubMed1985

Amine transport into chromaffin ghosts. Kinetic measurements of net uptake of biologically and pharmacologically relevant amines using an on-line amperometric technique.

The kinetic parameters for net transport of dopamine, epinephrine, norepinephrine, 5-hydroxytryptamine, S alpha-methyldopamine, R alpha-methyldopamine, and 1R,2S alpha-methylnorepinephrine into highly purified bovine chromaffin ghosts were determined using an on-line amperometric technique. Chromaffin ghosts devoid of endogenous amines were formed from lysis of chromaffin granules under hypotonic conditions, extensive washing of the scattered membranes, followed by resuspension in iso-osmotic media and overnight dialysis. When chromaffin ghosts formed so as to generate and maintain a large delta pH were suspended in 185 mM KCl, 10 mM Hepes at pH 7.0, 37 degrees C, the addition of MgATP resulted in rapid acidification of the intravesicular space, which was maintained at pH 6.0 (+/- 0.1) for over 30 min. Kinetic net amine transport was subsequently measured with a glassy carbon electrode. The initial rates of uptake were found to follow Michaelis-Menten kinetics. Computer based statistical analysis of the data using distribution-free procedures yielded Km (and V) values as follows: in microM (nmol X mg protein-1 X min-1) dopamine, 16.2 (14.0); R-norepinephrine, 32.5 (12.9); R-epinephrine, 35.1 (15.2); 5-hydroxytryptamine, 4.7 (5.1); S alpha-methyldopamine, 17.7 (11.2); R alpha-methyldopamine, 44.2 (9.9); 1R,2S alpha-methylnorepinephrine, 76.5 (12.5). The physiologic and pharmacologic implications of these kinetic parameters are discussed.

Adrenal Medulla↗
PubMed1985

Myocardial oxygen consumption with isoproterenol versus calcium chloride in hypocalcemic ventricular failure in dogs.

In 30 dogs on right heart bypass we compared the effects of isoproterenol with those of calcium chloride on myocardial oxygen consumption and on left ventricular function in the setting of ventricular depression produced by ionized hypocalcemia. In 22 dogs (Groups A and B) either isoproterenol or calcium chloride was infused, left ventricular function curves were generated, and end-diastolic pressure vs segment length plots were obtained. In 8 dogs (Group C), with initial hypocalcemia, both isoproterenol and calcium chloride were infused separately in random order to produce an equal decrease in left ventricular end-diastolic pressure at constant mean aortic pressure, heart rate, and cardiac output. Myocardial oxygen consumption and indices of left ventricular function were obtained. In Groups A and B, both drugs, when administered to the ventricle depressed by hypocalcemia, displaced left ventricular function curves upward and to the left. Left ventricular stroke work at constant left ventricular end-diastolic pressure increased (from 13.0 +/- 1.3 to 31.2 +/- 2.3 g X m for isoproterenol; from 13.9 +/- 2.5 to 32.5 +/- 2.5 g X m for calcium chloride). In Group C, there were no significant differences between left ventricular end-diastolic pressure, end-diastolic internal diameter, myocardial oxygen consumption, or peak left ventricular dP/dt in the hypocalcemic periods preceding isoproterenol and calcium chloride infusion. When the two drugs caused matched decreases in left ventricular end-diastolic pressure (-7.4 +/- 0.5 cm H2O for isoproterenol; -7.3 +/- 0.8 cm H2O for calcium chloride) there were similar decreases in end-diastolic internal diameter. However, isoproterenol was associated with a significantly greater (P less than 0.001) myocardial oxygen consumption (13.7 +/- 0.4 ml X 100 g-1 X min-1) than calcium chloride infusion (11.9 +/- 0.4 ml X 100 g-1 X min-1), as well as a greater peak left ventricular dP/dt (P less than 0.005).

Animals↗