Binding characteristics and analgesic activity of D-ALA2-Leu5-enkephalin chloromethyl ketone

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin (DALECK) was synthesized and its potency was tested in competing for 3H-naloxone binding sites and inducing analgesia. It was established that the compound is a potent affinity reagent at alkaline pH, blocking selectively and irreversibly the high-affinity (KD less than 1 nM) binding site. Intracisternally given DALECK showed a long-lasting, dose-dependent antinociceptive effect in the rat tail-withdrawal test. This could be completely antagonized by naloxone administration showing the reversible nature of DALECK in this in vivo assay. It is suggested that DALECK binds reversibly to the morphine receptor which mediates analgesia but irreversibly to the enkephalin receptor, the function of which remains to be elucidated.     

Simulation of rhythmic nervous activities

Zusammenfassung Die überschwellige Dauererregung mehrerer Jenik-Modellneurone würde bei jedem einzelnen zu einer ununterbrochenen Dauerentladung führen. Auf Grund des hier vorgestellten Verschaltungsprinzips der sog. Zyklischen Hemmung sind die Elemente jedoch über Hemmungsleitungen in zyklischer Weise miteinander verkoppelt, wodurch zeitlich definierte, periodisch sich wiederholende, gegenseitige Erregungsunterdrückungen erreicht werden. Das heißt: trotz gleichförmiger Eingangserregung zeigen die Neuronenmodelle am Ausgang ein burst-artiges Entladungsmuster.Der für einen solchen Burst-Generator endlicher Folgefrequenz notwendige Verzögerungsmechanismus ist dadurch gegeben, daß die Modellneurone nach erfolgter Hemmung eine ausreichend lange Erholphase durchlaufen müssen, bevor sie wieder in den aktiven Entladungszustand gelangen. Die Länge dieser Erholphase ist abhängig von den Parametern des Erregungs- und Hemmeinganges (Frequenz, Amplitude, PSP-Zeitkonstante), also von außen steuerbar.Zwei verschiedene Typen von zyklischen Netzwerken werden untersucht. In den einfachen Netzwerken werden Ausgangs- (Erregungs-) und Zwischen- (Hemmungs-) Nervenzelle durch ein und dasselbe Modellneuron repräsentiert. Beliebig viele, jedoch mindestens 3 Einzelelemente umfaßt ein solcher Burst-Generator (N3), wobei jedes Element in Hemmrichtung maximal bis zu M _maxN–2 Nachbarelemente hemmend beeinflussen kann. Diese streng rotationssymmetrische Hemmungsverschaltung garantiert das charakteristische rhythmische Ausgangsmuster der periodischen Erregungsumläufe, indem die Modellneurone entgegen dem Hemmrichtungssinn nacheinander in Bursts entladen.Im Unterschied zu den einfachen Netzwerken wird — als mögliche Annäherung an die physiologischen Gegebenheiten — in den sog. komplexen Netzwerken der Hemmeinfluß jedes Ausgangselementes über ein eigenes Zwischen- (Hemmungs-) Element ausgeübt. Die Vielfalt der Ausgangs-Zeitmuster kann dadurch erheblich gesteigert werden.Die Ausgangsmuster beider Netzwerktypen können bei gegebener Struktur als Funktion der Erholphase errechnet werden. Die Nützlichkeit dieser Netzwerke als biologisches Modell liegt vielleicht gerade darin, daß die das Zeitverhalten bestimmende Erholphase von den Eingangsgrößen direkt gesteuert wird, d. h. die Werte der Eingangsparameter werden in eine Zeitgröße (der Länge der Erholphase) umgesetzt. Mit noch zusätzlicher zeitabhängiger und asymmetrischer Variation dieser Eingangsparameter an den Einzelelementen stellt ein derartiges Netzwerk ein vielseitiges und flexibles Steuerinstrument für die verschiedensten (periodischen) Vorgänge dar.Obwohl es aus der Biologie noch keinen direkten Beweis für das Vorhandensein einer derartigen Hemmungsverkopplung gibt, sind andererseits rückführende Hemmverbindungen und Neurone mit relativ langer Hyperpolarisationsphase bekannt. Im Rückenmark, Septum, Ammonshorn und Thalamus sind in der Literatur ähnliche Neuronenaktivitäten beschrieben, die unsere Modellvorstellung unterstützen.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).     

Stomatal resistance,leaf water potential and hydraulic resistance of sugar beet plants

Stomatal resistance (r_L) and leaf water potential (Υ_L), soil moisture and the course of meteorological factors were measured in irrigated and non-irrigated sugar beet canopies during three years. By means of the canopy water balance equation, theoretical analysis of observed dependencies of stomatal resistance upon leaf water potential was made. The changes of r_L were not induced by the change of (Υ_L) but by that of external and internal factors, (Υ_L) correlates with. Therefore the empirical dependence of stomatal resistance upon leaf water potential cannot be generalized.     

Xylanase Activity of Phanerochaete chrysosporium

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30°C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-β-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. β-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. β-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.     

Role of endothelium, oxygen and ionic milieu in the prostacyclin and thromboxane production of rat aortic tissue slices.

The present study was executed in order to get further data on the role of vessel wall constituents in prostanoid synthesis and on the effect of anorganic constituents on it. Prostacyclin and tromboxane production of rat aortic tissue slices with intact endothelium and after mechanical as well as chemical endothelium removal were studied. The effects of hypoxia and changes in the ionic milieu on the release of these prostanoids were also examined. The tissue slices were incubated in normal or in modified Krebs-Ringer solution, bubbled with 95% O2 and 5% CO2 (with the exception of the studies in hypoxic conditions). Prostacyclin and thromboxane release was determined by specific radioimmunoassay of the stable metabolites, 6-keto-PGF1 alpha and TxB2, from the incubation medium. 174 tissue samples obtained from 164 rats were studied. Mechanical removal of the endothelium increased prostacyclin production of the aortic segments about fivefolds from a basal rate of 52.9 +/- 19.4 ng/gr/min, while it had no significant effect on thromboxane release (basal rate 0.83 +/- 0.13 ng/gr/min). Treating the endothelium with 1.0 M HCl almost totally suppressed prostacyclin release. Lowering the partial oxygen tension of the incubation medium significantly decreased the production of prostacyclin, while release of TxB2 somewhat increased. Increasing the Ca2+ concentration of the medium between 0-5 mM the release of prostacyclin was augmented and the release of thromboxane was diminished. Potassium free medium caused a very large increase in prostacyclin release of the tissue slices. The results show that release of vasoactive prostanoids from isolated rat aortic wall is dependent not exclusively on the endothelium and that various methods of endothelium removal may have distinct influences on prostacyclin and thromboxane productions. The changes in anorganic constituents of the surrounding medium could massively affect prostacyclin and thromboxane production of rat aortic tissue. The alternative effects of the above listed treatments on the release of prostacyclin and thromboxane from the rat aortic wall suggest the existence of different mechanisms in the control of the production of the two major prostanoids possessing opposite physiological effects.     

Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.