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Biomedical subjects

K A Fisher

Publications and source records attributed to K A Fisher.

14 recordsLinked to original sources
PubMed1992

Activation of proximal tubular Na(+)-H+ exchange by angiotensin II.

Stimulation of Na(+)-H+ exchange by angiotensin II (ANG II) was characterized in renal proximal tubular cells. Rabbit proximal nephron segments were incubated in the presence or absence of ANG II (5 x 10(-10) M), after which brush-border membrane vesicles (BBMV) were isolated and assayed for Na(+)-H+ antiporter activity using the acridine orange technique. Both the affinity (for sodium) and capacity of the carrier were elevated significantly (P less than 0.05) within 15 min of incubation with ANG II. To determine whether the stimulation of transport capacity involved a change in Na(+)-H+ antiporter density in the luminal membrane, binding of tritiated 5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) was measured in BBMV derived from control and ANG II-treated nephron segments, following maximal stimulation. This demonstrated a significant (P less than 0.05) increase in the maximal specific binding (Bmax) of [3H]MIA binding in the ANG II-treated group compared with control, of a magnitude sufficient to account for the observed change in maximal velocity (Vmax). The data indicate that the Vmax effect is caused by an apparent increase in the number (density) of active Na(+)-H+ carriers present in the luminal membrane. Finally, to test the possibility that the observed kinetic change involves an exocytic mechanism, the effect of colchicine on ANG II-stimulated antiporter activity was examined. The increase in Vmax due to ANG II was blocked by the addition of 0.5 mM colchicine to the incubation medium, whereas colchicine alone had no significant effect on the Vmax of Na(+)-H+ kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1979

Calcific stenosis of the porcine heterograft.

We have encountered two cases of late calcification of the porcine heterograft. A patient in chronic renal failure died of sepsis and endocarditis fifteen months after replacement of the mitral and tricuspid valves. At postmortem examination, both heterograft valves exhibited severe calcification and thrombosis. A second patient with rheumatic heart disease and sickle cell disease underwent mitral valve replacement for severe regurgitation. Thirty months later, cardiac catheterization revealed prosthetic valve stenosis. The valve was replaced successfully, and the excised heterograft exhibited severe calcification with restriction of leaflet motion. Although calcification of the porcine heterograft is known to occur in patients with infection or disorders of calcium metabolism, dysfunction of the heterograft is rare in our experience.

Adolescent↗
PubMed1978

Molecular orientation of bacteriorhodopsin within the purple membrane of Halobacterium halobium.

The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.

Bacteriorhodopsins↗
PubMed1978

Oriented adsorption of purple membrane to cationic surfaces.

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.

Adsorption↗
PubMed1977

Nephrotic proteinuria with pre-eclampsia.

During a retrospective study of 100 patients who underwent renal biopsy because of pregnancy complicated by hypertension, we found 19 patients whom proteinuria exceeded 5.0 Gm. per 24 hours and an additional eight patients in whom excretion ranged between 3.5 and 5 Gm. per day. Of these 27 patients, 23 had the kidney lesion of pre-eclampsia, and three of them had superimposed hypertensive changes in the vasculature. The remaining four had other renal diseases. We located and re-examined 10 of the 23 pre-eclamptic women, 12 to 104 (mean, 36) months after delivery. Serum creatinine levels were normal in all but one, who was discovered to have polycystic kidney disease. During the same time period, we located the records of six women who had heavy proteinuria during gestation but were normotensive. Thus, at our institution, pre-eclampsia is the most common cause of the nephrotic syndrome in pregnancy. The frequency of nephrotic proteinuria in pre-eclampsia appears higher than previously suspected, but, despite this fact, recovery was complete in most instances.

Creatinine↗
PubMed1977

Freeze-fractured purple membrane particles: protein content.

Optical diffraction and image reconstruction can be used to correlate the electron microscope image of the biological membrane with its electron density projection. Such correlation shows that a single purple membrane particle contains 9 to 12 protein molecules--63 to 84 transmembrane alpha helices--a complexity two to ten times greater than that previously suggested for membrane particles.

Bacteriorhodopsins↗
PubMed1977

Intramembranous particle distribution in human erythrocytes: effects of lysis, glutaraldehyde, and poly-L-lysine.

Freeze-fracture combined with quantitative electron microscopy of the intact human erythrocyte (RBC) and ghost revealed significant differences in their intramembranous particle coefficients. External (E) fracture-faces of unfixed ghost membranes were found to contain 40% fewer particles than those of intact unfixed RBC. The particle distribution of the intact RBC membrane depended on the use of glutaraldehyde fixation and glycerol cryoprotection. Whereas glutaraldehyde- and glycerol-treated cells disclosed 70% fewer E-face particles than did intact unfixed cells, poly-L-lysine-treated, intact, unfixed RBC showed no such differences. Treatment with a combination of poly-L-lysine and glutaraldehyde, however, increased the amount of E-face particles while reducing those of the protoplasmic (P) face. The poly-L-lysine effect varied with its concentration and was unaffected by previous application of neuraminidase. Nor did the lectin phytohemagglutinin induce particle rearrangement in intact cells. Our data demonstrate that the processes of glutaraldehyde fixation and glycerol cryoprotection modify the RBC membrane by decreasing the number of E-face particles present. In addition, the combination of poly-L-lysine and glutaraldehyde alters the affinity of some particles for one half of the membrane, suggesting that in freeze-fractured RBC, chemical bonds formed at the extracellular surface of the membrane can influence particle partitioning.

Aldehydes↗
PubMed1976

Prediction of azathioprine intolerance in transplant patients.

One cause of transplant rejection is curtailment of immunosuppressive therapy due to leucopenia. To determine those patients most apt to develop leucopenia due to azathiprine the granulocyte response to intravenous injections (i.v.) of hydrocortisone was evaluated in 10 patients who had rejected their grafts at least six month previously. 5 patients who had rejected their grafts with concomitant severe leucopenia had an inadequate response to hydrocortisone, while in the other 5, who had tolerated the drug, the response was similar to that of normal controls. Based on these observations, all the transplant candidates underwent the hydrocortisone stimulation test the results of which were correlated with their subsequent clinical course. All medical decisions were based on events other than the steroid test. 8 leucopenic patients underwent splenectomy. 6 improved their granulocyte response to hydrocortisone and tolerated azathioprine after transplantation. 2 patients who underwent splenectomy and an unoperated leucopenic man were unresponsive to the hydrocortisone test, did not tolerate azatioprine after transplantation and rejected their grafts. 4 candidates with normal responses to i.v. hydrocortisone received transplants uneventfully. In all 13 patients transplanted since the beginning of this study, the hydrocortisone test correctly predicted their tolerance to azathioprine.

Azathioprine↗
PubMed1976

Analysis of membrane halves: cholesterol.

Membrane splitting by freeze-fracture has been used as a preparative tool for chemical analysis of outer and inner "half"-membranes. In a previous report I showed that monolayers of human erythrocytes, bound to cationized glass, fracture nonrandomly, producing membrane fractions substantially enriched in outer or inner "halves." For the present study cells were used in quantities compatible with microanalysis. For quantitation the total amount of membrane present and the fractional enrichment of outer and inner "half"-membranes were determined. Cholesterol was examined by quantitative thin-layer chromatography modified to assay nanogram amounts. Comparison of lipids extracted from intact membranes with lipids from fractured membranes indicated that cholesterol was asymmetrically distributed across the plane of the membrane, more being present on the exterior side than on the interior side.

Cell Membrane↗
PubMed1976

Freeze-fracture autoradiography: feasibility.

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane with its chemical and functional characteristics. Within the limits of electron microscope autoradiographic resolution, FARG has the potential to detect the relative distribution of molecules in each half of the membrane and within the plane of the membrane. The use of radioisotopic labels in combination with freezing techniques requires minimal perturbation of the system being studied and may be suitable for the examination of substances which would be extracted or would diffuse during the normal fixation and embedding procedures used in standard electron microscope autoradiography.

Autoradiography↗
PubMed1976

Potassium secretion by colonic mucosal cells after potassium adaptation.

Recent studies have demonstrated that chronic potassium loading increases Na-K-ATPase specific activity in kidney tissue and suggest that this enzyme plays a role in renal potassium adaptation. Studies of fluid and electrolyte movement, potential difference (PD), AND Na-K-ATPase were performed in colon and jejunum of the rat in order to further characterize the relationship of Na-K-ATPase to potassium secretion. Experimental rats fed 2.6 meq K/gm diet for 7 days were compared to a control group fed 0.13 meq K/gm. In the colon, chronic potassium loading increased potassium secretion from 0.8 +/- 0.2 to 3.9 +/- 0.9 mueq/min per g tissue (P less than 0.01) and PD from 27 +/- 5.0 to 54 +/- 2.6 mV (P less than 0.001), lumen negative, as Na-K-ATPase increased from 5.0 +/- 0.5 to 11.4 +/- 1.0 muM Pi/mg protein per h (P less than 0.001). In contrast, there was no change in PD, potassium movement, or Na-K-ATPase in the jejunum of potassium-loaded rats. Colonic movement of water, sodium, and chloride was similar in the control and potassium-loaded rats. These results indicate that increased Na-K-ATPase is associated with both increased PD and increased potassium secretion in the colon and provide additional evidence suggesting that Na-K-ATPase may be important in the control of transepithelial potassium movement.

Action Potentials↗
PubMed1975

"Half" membrane enrichment: verification by electron microscopy.

Membranes of intact erythrocytes bound to polylysine-treated glass fracture nonrandomly when covered with thin copper and frozen. Electron microscopic examination of the glass side reveals extensive areas of outer "half" membrane (B face) and of the copper side, inner "half" membrane (A face). This technique allows the ultrastructural examination of square-centimeter areas of fractured membrane and the chemical analysis of these membrane "halves".

Cell Membrane↗
PubMed1975

Dissociation of Na-K-ATPase specific activity and net reabsorption of sodium.

Prevoius studies have suggested that the increase in specific activity of Na-K-ATPase in renal tissue during treatment with glucocorticoids occurred as a result of aconcurrent rise in net tubular reabsorption of sodium. Since recent data have indicated a specific effect of glucocortiocoids on epithelial cells, experiments were performed to determine whether enzyme activity and net sodium reabsorption could be dissociated. Evidence is provided demonstrating that base-line specific activity of Na-K-ATPase in rat renal cortex and outer medulla does not correlate directly with net sodium reabsorption since enzyme activity did not change after a chronic reduction in glomerularfiltration rate and the rate of sodium reabsorption. Further studies showed a markedrise in Na-K-ATPase after 4 days of treatment with methylprednisolone despite a fall in sodium absorption. These results suggest a direct effect of glucocorticoids onrenal Na-K-ATPase and illistrate the difficulty in assigning a transport role tothis enzyme from the correlation of specific activity with rates of net electrolyte transport.

Adenosine Triphosphatases↗