The influence of non-specific binding on scatchard analysis used in insulin/receptor binding studies with erythrocytes

Conclusions By equilibrium binding studies it could be demonstrated that erythrocytes have been saturated with specifically bound insulin in the order of 0.10–0.20 nmol/l with 4×10¹² erythrocytes/l. From this an insulin receptor number of 15–30 receptors/normal erythrocyte could be calculated.By the current results we got evidence that in the insulin/erythrocyte receptor system the terminal part of the Scatchard plot was artefactual and might not be used for the calculation procedures because it is strongly influenced by non-specific effects. Because of the fluid change from the specific to the non-specific character of binding we propose to use total ligand concentrations only up to 5 nmol/l with 4×10¹² erythrocytes/l for the investigation of specific binding on erythrocyte insulin receptors.By focussing on the initial part of the Scatchard plot curvilinearity disappeared and the character of this hormone/receptor system was reduced to one binding site with one characteristic affinity constant and a receptor concentration of one characteristic order.

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Der Einfluß der unspezifischen Bindung bei Anwendung des Scatchard Plots zur Untersuchung der Insulin/Receptor-Wechselwirkungen an Erythrocyten

     

Decreased insulin binding to mononuclear leucocytes and erythrocytes from dogs after S-Nitroso-N-Acetypenicillamine administration

Background

Nitric oxide (NO) and oxygen free-radicals play an important part in the destruction of beta-cells in auto- immune diabetes although the precise mechanism of interaction is still not known. This study was designed to examine any possible diabetogenic effect of NO by investigating any differences in cellular binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes of dogs treated with the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and controls treated with captopril.

Results

The result obtained showed decreased binding of insulin to its receptor on the cell membranes of erythrocytes and mononuclear leucocytes. Mononuclear leucocytes from SNAP-treated dogs had decreased ability to bind insulin (16.30 ± 1.24 %) when compared to mononuclear leucocytes from captopril-treated controls (20.30 ± 1.93 %). Similar results were obtained for erythrocytes from dogs treated with SNAP (27.20 ± 1.33 %) compared with dogs treated with captopril (34.70 ± 3.58 %). Scatchard analysis demonstrated that this decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with mononuclear leucocytes of SNAP-treated dogs having 55 % less insulin receptor sites per cell compared with those of captopril-treated controls (P < 0.05). Average affinity and kinetic analysis revealed a 35 % decrease in the average receptor affinity, with mononuclear leucocytes from captopril-treated controls having an empty site affinity of 12.36 ± 1.12 × 10^-8 M^-1 compared with 9.64 ± 0.11 × 10^-8 M^-1 in SNAP-treated dogs (P < 0.05).

Conclusion

These results suggest that acute alteration of the insulin receptor on the membranes of mononuclear leucocytes and erythrocytes occurred in dogs treated with S-nitroso-N-acetylpenicillamine. These findings suggest the first evidence of the novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus.     

Untersuchung von Membranelektroden mit Dibenzo-18-krone-6 und Derivaten

Zusammenfassung Homogene Membranelektroden mit Dibenzo-18-krone-6(I) und den Derivaten II, III, IV wurden untersucht. Die Elektroden sind selektiv für Cesium: Cs>Rb>K>Na>Li. Sie sprechen bei Aktivitäten >10^–3 mol/l geringfügig auf Anionen an. Ihre Lebensdauer beträgt maximal drei Wochen.I–III weisen in den Elektrodenmembranen eine geringe Löslichkeit auf. Dies beeinträchtigt besonders die Potentialbildung der Elektroden mit Festmembranen. IV löst sich in den Membranen besser als I–III, ist jedoch weniger selektiv für Cesium.

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Examination of membrane electrodes based on dibenzo-18-crown-6 and derivatives

Summary Homogeneous membrane electrodes with dibenzo-18-crown-6(I) and its derivatives II, III, IV have been examined. The electrodes are selective for cesium: Cs>Rb>K>Na>Li. They respond insignificantly to anions having an activity of >10^–3 mol/l. Their life-times run to three weeks.I to III are slightly soluble in the electrode membranes. This interferes especially with the formation of potentials of the electrodes with solid-membranes. IV has a higher solubility in the membranes than I to III, but is less selective for cesium.

     

FTIR-ATR-spectroscopic analysis of Bis-Crown ether based PVC-membranes

Summary To get more information on the phase boundary and bulk membrane processes, the short and long time behaviour of potassium selective solvent polymeric membranes was studied. In parallel with the measurement of the dynamic response of model membranes, in situ spectroscopic measurements applying the Attenuated Total Reflection (ATR) technique in the infrared region, were carried out in solutions containing anions of different lipophilicity (Cl^– and SCN^–). Thin membranes (thinner than the penetration depth of IR beam) coated on the germanium reflection element by the dipping technique show no time dependence of the SCN bands in contact with KSCN solution of different concentrations, while the diffusion process of species can be monitored in thick membranes as it was shown in a time resolved study. The extremely short response times of ionophore based solvent polymeric membranes (determined under appropriate experimental conditions in this work) imply that the fundamental potential determining step of the overall response of this type of electrodes is a phase boundary process. Thus the slow inner membrane diffusion process followed by IR spectroscopy on the molecular level may affect only the long-term membrane behaviour.

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FTIR-ATR-spektroskopische Analyse von Bis-Kronenether-PVC-MembranenIII. Spektroskopische und elektrochemische in situ-Methoden zur Untersuchung von OberflÄchen- und Bulk-Membran-Prozessen

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Part II: see [4]

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Dedicated to Prof. Dr. W. Fresenius on the occasion of his 75th birthday     

Neutral carrier based silicone rubber membranes for H3O+, K+, NH4 + and Ca2+ selective electrodes

Summary Silicone rubber is a universal membrane matrix for neutral carrier based ion-selective electrodes. Carrier antibiotics as well as synthetic ionophores yield sensors with selectivities that are comparable or even higher than those of corresponding poly(vinyl chloride) (PVC) membrane systems. The permeability of silicone rubber membranes for gas molecules is about ten times higher than the permeability of PVC membranes. In contrast, the electrical conductivity is by a factor of 10–100 lower.

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Auf neutralen Carriern beruhende Silikongummimembran-Elektroden mit Selektivität für H₃O⁺, K⁺, NH₄ ⁺ und Ca²⁺

Zusammenfassung In ionenselektiven Elektroden auf der Basis von neutralen Carriern läßt sich Silikongummi als universelle Membranmatrix einsetzen. Entsprechende Sensoren sind sowohl mit ionenselektiven Antibiotika als auch mit synthetischen Carriern realisiert worden. Ihre Selektivitäten übertreffen teilweise jene von entsprechenden PVC-Membranen. Die Permeabilität der Silikongummimembranen für Gasmolekel ist ungefähr zehnmal höher als jene von PVC-Membranen. Andererseits ist die elektrische Leitfähigkeit um einen Faktor 10–100 niedriger.

     

Lipogenesis stimulatory and inhibitory activities of the insulin mediators

Incubating plasma membranes prepared from pig liver with varying concentrations of insulin (50-1000 microU/ml) resulted in the release of at least two insulin chemical mediators. One of them was fraction 1 of insulin mediator (M. W. 3700-4000 daltons) which had a significant lipogenesis-stimulating activity. The other was fraction 2 of insulin mediator (M. W. about 1000 daltons) which exhibited a lipogenesis-inhibitory activity. The ratio of yield between the two mediators produced from the membranes was not only dependent on the concentration but also on the potency of insulin and its analogs added. The result showed that there was more production of fraction 2 than fraction 1 with the inducer at low concentration (100 microU/ml), while the production of fraction 1 from the plasma membranes incubated with high concentration of insulin (300 microU/ml) was higher than fraction 2. On the other hand, insulin and its analogs which have different biological activities and receptor binding activities have been used to induce the insulin mediators. The results obtained were similar to those mentioned above. This suggested that the generation of the mediators was dependent on the biological potences but not the binding activities.     

Cholesteryl sulfate in meconium: Inhibitory action on rabbit anti-M and anti-N antisera

A lipid present in meconium and capable of inhibiting the agglutination of human M and N erythrocytes by rabbit anti-M and anti-N antibodies has been isolated and identified as cholesteryl sulfate. The reaction of human anti-A and anti-B antisera with the respective human red blood cells is not impaired by cholesteryl sulfate. Cholesteryl hemisuccinate has been found to be an hemagglutination inhibitor qualitatively similar to but less active than cholesteryl sulfate.

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Cholesterylsulfat in Mekonium: Hemmende Wirkung auf Kaninchen anti-M und anti-N Antiseren

Zusammenfassung Eine lipophile Substanz, welche die Agglutination humaner Erythrocyten durch Kaninchen anti-M und anti-N Antiseren inhibiert, konnte aus Mekonium isoliert und als Cholesterylsulfat identifiziert werden. Die Reaktion humaner anti-A und anti-B Antiseren mit den jeweiligen A und B Erythrozyten wurde durch Cholesterylsulfat nicht beeinflußt. In bezug auf die Agglutinationshemmung verhält sich der Monocholesterylester der Bernsteinsäure ähnlich wie der der Schwefelsäure, ist jedoch erst in höherer Konzentration wirksam.

     

Na+-Selektive Monensin-PVC-Membranelektrode mit Festableitung

Zusammenfassung Eine Monensin-PVC-Membran zur Applikation an Festkontakt-Sensoren wurde entwickelt. Die Meßketten zeigen in Abhängigkeit von der Na⁺-Ionenaktivität ein Nernst-gemäßes elektromotorisches Verhalten. Für die Störionen aus der Reihe der Alkali- und Erdalkaliionen sind die Selektivitätskoeffizienten auch als Funktion ihrer Ionenradien angegeben.

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Na⁺ selective monensin PVC membrane electrode with solid contact

Summary A Monensin PVC membrane has been developed for application to solid contact sensors. Depending on the Na⁺ ion activity, the electrochemical cells show a Nernstlike electromotive behaviour. For interfering ions of the alkaline and alkaline earth series, the selectivity coefficients are also given as a function of their ion radii.

     

Zum Auftreten von Asymmetriepotentialen in der Potentiometrie

Zusammenfassung Bei ionenselektiven Membranelektroden treten häufig Asymmetriepotentiale auch dann auf, wenn die Membranen nominell symmetrisch sind. Die Existenz solcher Potentiale wird unter Zuhilfenahme einfacher thermodynamischer und kinetischer Vorstellungen interpretiert. Weiterhin werden frühere Beobachtungen diskutiert, die mit der Vorstellung, daß sich die Potentialsprünge an der inneren und äußeren Oberfläche einer Membran unabhängig voneinander ausbilden, nicht vereinbar sind.

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Some aspects of asymmetry potentials in potentiometry

Summary Ion-selective membrane electrodes frequently develop asymmetry potentials even when the membranes are nominally symmetrical. The existence of such potentials is interpreted in terms of simple thermodynamic and kinetic ideas. Furthermore, former findings are discussed which argue strongly against the conception that the potential jumps at the inner and outer surface of a membrane arise independently.

     

Porous imprinted polymer membranes prepared by phase separation in compressed liquid CO<Subscript>2</Subscript>

Phase separation of poly(acrylonitrile–co-methacrylic acid) in compressed liquid CO₂ resulted in formation of a porous imprinted membrane which preferentially adsorbed uracil (URA). The cross-section of the membrane was observed by SEM, which revealed its porous structure. The mechanical strength of the membrane indicated formation of a rigid matrix with high tensile strength (4.4 N mm⁻²). The imprinted membranes bound highly selectively to URA (12.8 μmol g⁻¹) but binding to dimethyluracil (DMURA), thymine, and cytosine was less (0.7, 0.8, and 0.9 μmol g⁻¹, respectively). When DMURA was similarly used to prepare an imprinted membrane in liquid CO₂ there was less binding of DMURA to the imprinted membrane obtained. The URA-imprinted membranes were evaluated by IR spectroscopy before and after URA extraction. The results indicated that hydrogen bonding was the mechanism of binding of URA to the imprinted membrane. Competitive binding studies were performed with binary mixtures of URA and its analogues. The URA-imprinted membrane enabled good separation of URA from cytosine, DMURA, and thymine, with separation factors of 3.0, 3.8, and 2.5, respectively. It was confirmed that the compressed liquid CO₂ contributed to efficient formation of template substrate sites in the URA-imprinted membrane.     

Surface modification with nitrogen-containing plasmas to produce hydrophilic,low-fouling membranes

Nitrogen-based plasma systems such as N₂, NH₃, Ar/NH₃, and O₂/NH₃ were used to modify microporous polyethersulfone membranes. Treatments were designed to alter the surface chemistry of the membranes to create permanently hydrophilic surfaces. Contact angle measurements taken initially, as well as 1 year post-treatment confirmed that treatments using O₂/NH₃ plasmas (with a 5:3 gas flow ratio) were successful in achieving our designed goals. Analyses by FT-IR and XPS established the incorporation of NH_x and OH species in the PES membranes. Moreover, the plasma penetrates the thickness of the membrane, thereby modifying the entire membrane cross-section. Optical emission spectroscopy studies of excited state species present in the modifying gases revealed the presence of OH^*, which was not present in a 100% ammonia plasma, suggesting OH^* must play a critical role in the membrane modification process. Investigations using bubble point analysis, differential scanning calorimetry, and scanning electron microscopy demonstrate there is no damage occurring under these specific treatment conditions. The usefulness of this treatment is revealed by increased water flux, reduced protein fouling, and greater flux recovery after gentle cleaning when compared to an untreated membrane.     

Ionenselektive Coated Glass-Elektroden

Zusammenfassung Na⁺-, K⁺-, NH ₄ ⁺ - und Ca²⁺ selektive coated glass-Elektroden wurden entwickelt, bei denen eine ionenselektive Carrier-PVC-Membran auf eine ionenselektive Glasschicht aufgebracht wird, die über einen Innenelektrolyten mit der Ableitelektrode in Verbindung steht. Die Selektivität wird durch die aufgebrachte Carrier-PVC-Membran bestimmt, die zusätzlich Weichmacher zur Optimierung der Eigenschaften enthält.

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Ion-selective coated glass electrodes

Summary Na⁺, K⁺, NH ₄ ⁺ and Ca²⁺ selective electrodes have been developed by coating the ion-selective glass layer with a carrier PVC membrane. The glass is in contact with the conducting electrode through an inner electrolyte. The selectivity is determined by the PVC membrane, containing neutral carriers or electrically charged ligands and additionally plasticizers for optimizing the properties.

Dir Arbeit wird vom Bundesministerium für Forschung und Technologie der Bundesrepublik Deutschland gefördert.Prof. Dr. W. Simon stellte freundlicherweise die synthetischen neutralen Carrier für Na⁺ und Ca²⁺ zur Verfügung.     

Ansprechzeiten an Festk?rpermembranelektroden (I?, Cl?) bei schnellen Konzentrations?nderungen in str?menden L?sungen

Zusammenfassung Es wurde der Einfluß verschiedener experimenteller Parameter, wie Konzentrationsverhältnis, Endkonzentration, Richtung der Konzentrationsänderung, Konditionierung und Oberflächenbehandlung, Temperatur, Ionenstärke und Viskosität auf die Ansprechzeiten von Festkörpermembranelektroden untersucht.

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Response time of solid-state membrane electrodes (I^–, Cl^–) under fast changes of concentration in streaming solutions

Summary The influence of different experimental parameters like concentration ratio, final concentration, direction of concentration change, conditioning and surface treatment of the electrodes, temperature, ionic strength and viscosity on the response time of solid-state membrane electrodes is described.

Die vorliegende Untersuchung wurde in dankenswerter Weise durch Mittel der Deutschen Forschungsgemeinschaft und des Verbandes der chemischen Industrie unterstützt.     

Isolation and cleanup of organophosphorus insecticides and their oxones from animal tissues

Summary A simple isolation and cleanup procedure for the determination of organophosphorus insecticides and their oxones in animal tissues has been developed. This procedure unites partly extraction of organophosphorus pesticides and their oxones with cleanup by column chromatography. Most polar coextracts remain on the column and in its further processing the eluate does not tend to form emulsion. The process has been studied on model mixtures of ³²P- and ¹⁴C-labelled pesticides with tissues as well as after application of pesticides to white rats. The average recovery of the method is 90.5%, with a standard deviation of ±5.1%.

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Isolierung und Reinigung von Organophosphorinsecticiden und ihren Oxonen aus tierischen Gewehen

Zusammenfassung Ein einfaches Isolations- und Reinigungsverfahren wurde ausgearbeitet. Die Extraktion der Insecticide aus einer Homogenatsäule, die aus Kieselgel und Tiergewebe besteht, umfaßt zum Teil auch die Reinigung der Extrakte, da die polaren Verbindungen an die Säule gebunden bleiben. Der Mittelwert der Ausbeute des Verfahrens wurde im Modellversuch mit ¹⁴C- und ³²P-markierten Insecticiden zu 90,5% festgestellt. Die Standardabweichung beträgt ± 5,1%.

     

Anab initio investigation of rotation and inversion barriers in formylphosphine

Rotation about N–CO bonds in amides has been extensively investigated, but a corresponding barrier to rotation about the P–CO bond in an acylphosphine has yet to be observed. In the present 4-31Gab initio study of formylphosphine, rotation barriers of 9.6 and 13.5 kJ mol^–1 and a phosphorus pyramidal inversion barrier of 108.0 kJ mol^–1 are predicted. A comparison of STO-3G and STO-3G^* barriers suggests that polarization functions are not needed to describe rotation in this system.

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Eine ab initio Untersuchung von Rotations- und Inversions-Barrieren in Formylphosphin

Zusammenfassung Die Rotation um N–CO-Bindungen in Amiden wurde bisher intensiv untersucht, eine entsprechende Rotationsbarriere für Drehungen um die P–CO-Bindung in Acylphosphinen wurde jedoch nicht beobachtet. Eine 4-31Gab initio-Untersuchung an Formylphosphin ergibt Rotationsbarrieren von 9,6 und 13,5 kJ mol^–1 und eine pyramidale Inversionsbarriere von 108,0 kJ mol^–1 als Voraussage. Ein Vergleich der STO-3G und STO-3G^* Barrieren legt nahe, daß Polarisationsfunktionen für die Beschreibung der Rotation in diesen Systemen nicht nötig sind.

     

A sensitive bioluminescent assay of alcohol dehydrogenase in human serum and tissue extracts

Conclusions With the aid of a sensitive bioluminescence assay, alcohol dehydrogenase activity can be measured in the plasma of chronic alcoholics and patients with other drug-related liver-disorders. Suitable reagents for the assay are commercially available but must be compared regarding sensitivity and stability before routine determinations are introduced in clinical practice.

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Bestimmung der Alkoholdehydrogenase-Aktivität in menschlichem Serum und Gewebeextrakten mit Hilfe einer empfindlichen Bioluminescenz-Methode

     

Action of surface-active substances on biological membranes I. Effect of chemical modification of membranes on hemolysis of erythrocytes by sodium alkyl sulfates

Summary The hemolytic action of a homologous series of sodium alkyl sulfates (C ₈ toC ₁₅) on dog and human erythrocytes was measured. The influence of trypsin, pronase and neuraminidase treatments on the lytic resistance of human red cells was studied. A new approach evaluating the hemolytic activity of ionic detergents is expressed as a ratio of a concentration providing 50% lysis to its CMC-value. The results obtained are examined for their bearing on the use of surface-active agents as a means to study the structure organization of biological membranes.

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Zusammenfassung Es wurde die hämolytische Wirkung homologer Reihen von Natriumalkylsulfaten an Erythrozyten von Mensch und Hund gemessen. Um die hämolytische Aktivität der ionogenen Detergenzien zu charakterisieren, wurde das Verhältnis der Konzentration, welche eine 50%-ige Lyse bewirkt, zum cmc-Wert gewählt.

     

Radiometrische Schwefelbestimmung nach dem Entwicklungsverfahren

Zusammenfassung Unter Beibehaltung des Entwicklungsverfahrens wird eine Schwefelbestimmung in unlegiertem Stahl, Roh- und Gu\eisen ausgearbeitet, die sich durch Schnelligkeit und Sicherheit auszeichnet. Die Absorptionslösung wird durch Zusatz von ¹¹⁵Cd markiert und die RestaktivitÄt der Lösung nach Abtrennung von CdS bestimmt. Minderbefunde, die sich durch die grö\ere Löslichkeit von CdS in der essigsauren Absorptionsflüssigkeit ergeben, können durch einen experimentell ermittelten Korrekturwert eliminiert werden.

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Summary Based on the escapement method a rapid and safe determination of sulphur in non-alloy steel, pig iron and cast iron has been worked out. The absorbent solution is labeled by addition of ¹¹⁵Cd and residual activity is measured after separation of CdS. Tow low results may be found due to the increased solubility of CdS in the acetic acid absorbent solution, but can be improved by an experimental correction value.

     

Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

1H and13C NMR studies on lanthanide complexes with proline and hydroxyproline

The formation and the mode of coordination of rare earth (Y, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu) complexes with proline and hydroxyproline have been investigated by¹H and¹³C NMR spectral techniques. It has been established that the nitrogen and the carboxyl group of the ligands are involved in complexation, and that the OH^– group of hydroxyproline does not participate in coordination.

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¹H und¹³C NMR Untersuchungen an Lanthanid-Komplexen mit Prolin und Hydroxyprolin

Zusammenfassung Die Bildung und die Koordination von seltenen Erden (Y, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu) in Komplexen mit Prolin und Hydroxyprolin wurden mit Hilfe von¹H- und¹³C-NMR-Spektroskopie untersucht. Es wurde festgestellt, daß in den Komplexen der Stickstoff und der Carboxylsauerstoff der Liganden koordinieren. Die OH^–-Gruppe von Hydroxyprolin nimmt keinen Anteil an der Koordinierung.