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Biomedical subjects

E A Johnson

Publications and source records attributed to E A Johnson.

At least 163 records · Page 9Linked to original sources
PubMed1986

Physicochemical purification and immunological characteristics of ductal carcinoma antigen.

Ductal carcinoma antigen (DCA), as recognized by monoclonal antibody (MAb) F36/22, was purified in high yield and to homogeneity from malignant effusions by means of physicochemical techniques. Fractionation procedures were monitored by immunoenzymometric assays. Preparations purified over 2,000-fold were obtained through acid-extraction, lectin-chromatographies and gel filtration steps. Purified antigen demonstrated a single component upon electrophoretic examination. This material had a high molecular weight and high immunoreactivity. Neuraminidase treatments failed to perturb the antigenicity of the component, indicating an absence of sialic acid residues at the antibody-combining site. Further, extensive proteolytic digestion effected the release of heterogeneous glycopeptides which retained immunologic activity, suggesting the presence of carbohydrate at the active site. Immunologically, DCA was compared with other tumor-associated mucin-like glycoproteins which have been detected in the circulation of patients. Results indicated no cross-reactivity between DCA and other mucins, including CA19-9 and CA-125. Further, no antigenic relationship was noted for purified carcinoembryonic antigen (CEA), also a heavily-glycosylated structure. These data thus suggest that immunological monitoring of disease may be approached using a selected panel of antigenically distinct reagents.

Adenocarcinoma↗
PubMed1986

Lead precipitation: an aid to separation of dermatan and mucokeratan sulfates from glycosaminoglycan mixtures.

Dermatan sulfate with a high iduronate content may be precipitated selectively from solution with lead acetate at neutral pH. Other glycosaminoglycans, including dermatan sulfate with appreciable glucuronate content, are in general not precipitated. Both dermatan sulfate and mucokeratan sulfate are precipitable by the alkaline copper procedure, so that prior removal by lead acetate of some or all of the dermatan sulfate from a mixture of glycosaminoglycans permits concentration of mucokeratan sulfate. This eases considerably the detection and isolation of this recently identified glycosaminoglycan.

Animals↗
PubMed1986

Improving the operating room to increase revenues.

Hospitals often overlook the potential to increase revenues by improving operating rooms and attracting additional surgery. Administrators should respond to surgeons' dissatisfaction with services despite limitations and ambiguities imposed by the surgical suite's chain of command. Directors of surgery can overcome formal authority problems by establishing informal relationships with the chiefs of surgery and anesthesia. Monthly reports to administration on quality and performance standards can avoid difficulties and prompt timely solutions. Satisfied surgeons will increase admissions by directing elective surgery patients to the hospital. Administrators can conduct interviews with all surgical personnel or use questionnaires to identify problems, potential new surgeons, and suggestions for improvement. Other tasks are to analyze each surgical section's functions, accumulate data by specialty and surgeon to plan daily schedules, evaluate organization and staffing, and evaluate instrumentation and supplies. Block scheduling and surgical acuity systems help establish controls in these areas. Feedback systems ensure that personnel remain sensitive to surgeons' needs. A nurse should be assigned to each surgeon to communicate information about patients, and surgeons should be encouraged to complete forms after each surgery to identify deficiencies in procedures. Other incentives for surgeons include favorable OR scheduling arrangements, purchase of special equipment, and package pricing for certain procedures.

Anesthesia Department, Hospital↗
PubMed1986

Reconstruction of transport currents during repolarization: biochemical basis.

A philosophy and approach is described for including enzyme-mediated transport currents in reconstructions of ion regulation and electrical activity in cardiac muscle. Data from physiological and biochemical experiments on isolated transport systems are combined with the principles of physical chemistry to construct mechanistic descriptions of the systems. These descriptions are then combined (unmodified) together with the results of morphological measurements on cells to reconstruct the behavior of the ion regulation system. Some results from a preliminary model of this type are described: calcium regulation by the plasmalemma, including sodium-calcium exchange, the calcium pump (ATPase), and a calcium leak. This subsystem is stable at physiological values of ion concentrations and transmembrane potential and the net flux through the leak is within the range determined experimentally. Under these conditions, most of the calcium entering the cell through the leak is shown to be restored by the calcium pump. From calculations with an action potential of arbitrary waveform, it is shown that sodium-calcium exchange can make a small, but measurable contribution to repolarization in the cardiac cell.

Computer Simulation↗
PubMed1985

Standardization of low molecular weight heparins: a collaborative study.

A collaborative study was carried out, in which eight laboratories each assayed eight low molecular weight (LMW) heparins against the International Standard (IS) for heparin. APTT assays and three types of anti-Xa method were used. The results of this study showed that: LMW heparins cannot be validly assayed against the IS by APTT or anti-Xa methods. Potencies of LMW heparins vs. the IS differed considerably between the four types of assay method used and also between different laboratories using the same type of method. Adoption of a single LMW heparin standard would improve validity, improve inter-laboratory variation, and largely abolish the differences between the three types of anti-Xa method. However, since calibration of a LMW heparin standard against the IS would give potencies that differ widely by the different assay methods, a single assay method such as the anti-Xa amidolytic, plasma, would need to be chosen for this calibration.

Antithrombins↗
PubMed1985

A minimum mechanism for Na+-Ca++ exchange: net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential.

Both simultaneous and consecutive mechanisms for Na+-Ca++ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca++ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na+ ions and one Ca++ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 3:1 Na+: Ca++ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(-FV/2RT). With reasonable values for the membrane density of the enzyme (approximately 120 sites micron 2) and an upper limit for the rate constants of both translocational steps of 10(5) . sec-1, satisfactory behavior was obtainable with identical binding constants for Ca++ on the two sides of the membrane (10(6) M-1), similar symmetry also being assumed for the Na+ binding constant (12 to 60 M-1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.

Animals↗
PubMed1985

Application of the principle of linked functions to ATP-driven ion pumps: kinetics of activation by ATP.

If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct participant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center.

Adenosine Triphosphate↗
PubMed1985

Thermodynamic and kinetic cooperativity in ligand binding to multiple sites on a protein: Ca2+ activation of an ATP-driven Ca pump.

Contrary to common belief, theoretical analysis does not predict any necessary relationship between cooperativity in the equilibrium binding of an ion to multiple binding sites on a protein and cooperativity in the kinetic activation of a reaction for which such binding is prerequisite. The sarcoplasmic reticulum Ca pump protein, for example, has two high-affinity binding sites for Ca2+, here considered to be nearly identical and independent. Equilibrium binding to these sites can be highly cooperative in spite of site-independence, as demonstrated by the well-known allosteric mechanism based on Wyman's principle of linked functions. We show in this paper that kinetic activation of the pump reaction cycle by binding of Ca2+ to these same sites can likewise be a cooperative function of Ca2+ concentration but that the criteria that determine cooperativity in the two situations are different. It is possible to observe kinetic cooperativity without concomitant cooperativity in equilibrium binding and vice versa. Application of these theoretical considerations to experimental data for the pump protein raises questions about the Ca2+ binding mechanism.

Animals↗
PubMed1985

Variable stoichiometry in active ion transport: theoretical analysis of physiological consequences.

Active ion transport systems with fixed stoichiometry are subject to a thermodynamic limit on the ion concentration gradients that they can generate and maintain, and their net rates of transport must inevitably decrease as this limit is approached. The capability to vary stoichiometry might thus be physiologically advantageous: a shift to lower stoichiometry (fewer ions pumped per reaction cycle) at increasing thermodynamic load could increase the limit on the supportable concentration gradient and could accelerate the rate of transport under high-load conditions. Here we present a theoretical and numerical analysis of this possibility, using the sarcoplasmic reticulum ATP-driven Ca pump as the example. It is easy to introduce alternate pathways into the reaction cycle for this system to shift the stoichiometry (Ca2+/ATP) from the normal value of 2:1 to 1:1, but it cannot be done without simultaneous generation of a pathway for uncoupled leak of Ca2+ across the membrane. This counteracts the advantageous effect of the change in transport stoichiometry and a physiologically useful rate acceleration cannot be obtained. This result is likely to be generally applicable to most active transport systems.

Adenosine Diphosphate↗
PubMed1985

Incorporation of membrane potential into theoretical analysis of electrogenic ion pumps.

The transport rate of an electrogenic ion pump, and therefore also the current generated by the pump, depends on the potential difference (delta psi) between the two sides of the membrane. This dependence arises from at least three sources: (i) charges carried across the membrane by the transported ions; (ii) protein charges in the ion binding sites that alternate between exposure to (and therefore electrical contact with) the two sides of the membrane; (iii) protein charges or dipoles that move within the domain of the membrane as a result of conformational changes linked to the transport cycle. Quantitative prediction of these separate effects requires presently unavailable molecular information, so that there is great freedom in assigning voltage dependence to individual steps of a transport cycle when one attempts to make theoretical calculations of physiological behavior for an ion pump for which biochemical data (mechanism, rate constants, etc.) are already established. The need to make kinetic behavior consistent with thermodynamic laws, however, limits this freedom, and in most cases two points on a curve of rate versus delta psi will be fixed points independent of how voltage dependence is assigned. Theoretical discussion of these principles is illustrated by reference to ATP-driven Na,K pumps. Physiological data for this system suggest that all three of the possible mechanisms for generating voltage dependence do in fact make significant contributions.

Biological Transport, Active↗
PubMed1985

Polycythemia vera, primary hyperparathyroidism, and dysplastic nevus syndrome: report of a case.

Polycythemia vera and primary hyperparathyroidism is diagnosed in a patient with dysplastic nevus syndrome. A 59-year-old white man is noted to have numerous pigmented lesions found predominantly over the upper aspect of the back and chest. A biopsy revealed the atypical nevi found in dysplastic nevus syndrome. Over a 13-year period, three superficial spreading malignant melanomas were excised. The patient subsequently had polycythemia vera and primary hyperparathyroidism develop. Atypical pigmented skin lesions were not seen in family members, although two family members died of metastatic malignant neoplasms, one from endometrial carcinoma and the second from prostatic adenocarcinoma.

Humans↗
PubMed1985

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.

Cations, Divalent↗
PubMed1984

Heparan sulphates from porcine intestinal mucosa. Preparation and physicochemical properties.

A series of fractions of heparan sulphate has been prepared from the glycosaminoglycuronans of porcine intestinal mucosa, in order to relate their physicochemical properties with their biochemical and biological activities. They fall into three classes which may be distinguished by differences in molecular weight distribution and in extent of sulphation. For heterogeneous polymers such as heparan sulphates purification and fractionation must be regarded as interchangeable terms, and some preparative procedures appear to have rather specific effects on the properties of samples isolated by their use. Such considerations apply particularly in the undemarcated boundary region between heparan sulphates and heparin, a region which includes certain 'high activity' heparin fractions of current biological and clinical interest.

Animals↗
PubMed1984

Immunoaffinity isolation of ductal carcinoma antigen using monoclonal antibody F36/22.

Monoclonal antibody (McAb) F36/22, raised against a human breast tumor line, identifies an antigen found in the circulation of cancer patients. Antigen was purified from malignant effusions using McAb-affinity chromatography followed by adsorption-desorption from immobilized wheat germ lectin. Electrophoretic analysis demonstrated the isolation of a single high mol. wt glycoprotein exhibiting an isoionic point near pH 4.2 and a density of approx. 1.45 g/ml. Although highly reactive with wheat germ lectin, a negligible or weak interaction was observed with concanavalin A, lentil and peanut agglutinin. The antigen was immune-precipitable, indicating the occurrence of multiple McAb-binding sites, and was resistant to heat and acid treatments. Antigenicity was not perturbed following protease or neuraminidase treatments, but was affected upon exposure to alkaline conditions. Taken together, these data suggest that McAb F36/22 recognizes a high mol. wt component occurring in circulation as a mucin-like glycoprotein.

Antibodies, Monoclonal↗
PubMed1984

Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae.

Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha regulon responsible for fermentative dissimilation of glycerol and DHA, was purified 120-fold to a final specific activity of 10 mumol X min-1 X mg of protein-1 at 30 degrees C. The enzyme, a dimer of a 53,000 +/- 5,000-dalton polypeptide, is highly specific for DHA (Km, ca.4 microM). Glycerol is not a substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate. Antibodies raised against E. coli glycerol kinase cross-reacted with K. pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.

Glycerol↗