Affinity chromatography purification of Pseudomonas aeruginosa exotoxin A on specifically linked NAD agarose.

The specific binding of P. aeruginosa exotoxin A to NAD was exploited for the rapid purification of the toxin. Affinity chromatography on a column of agarose-N6-(aminohexyl)carbamoylmethyl-NAD resulted in an enzymatically, biologically, and immunologically active purified toxin preparation. Other NAD-agarose resins were not efficient substrates for toxin purification.     

Chemical structure and properties of duck and goose fibrinogen

Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented.     

Calorimetric studies of flavin-binding proteins: FMN and FAD binding to hen egg riboflavin-binding proteins

Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 10⁵ and 10⁴ were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form.     

Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones.

The subfamily of guanine nucleotide-binding regulatory (G proteins) designated Gq has been shown to regulate the activity of phospholipase C by reconstitution. However, the role of these proteins in hormonal regulation of this activity has not been demonstrated. Two antisera were used in attempts to interrupt this pathway. Antiserum W082, developed against a peptide representing an internal sequence in alpha q, was specific for alpha q by immunoblots but did not recognize the native protein. Antiserum X384 was developed against a peptide representing the 12 amino acids of the common carboxyl termini of alpha q and alpha 11. It had a broader specificity for this subfamily of G protein alpha subunits and recognized the native proteins. Antiserum X384 specifically immunoprecipitated alpha q and its homologs from purified preparations and detergent extracts of membranes. Affinity-purified antibodies attenuated stimulation of phosphatidylinositide 4,5-bisphosphate hydrolysis by bradykinin, angiotensin, and histamine in membranes derived from NG108-15 cells, rat liver, and 1321N1 cells, respectively. Activation of the phospholipase C activity by guanosine 5'-3-O-(thio)triphosphate alone was also inhibited. Inclusion of the peptide to which the antisera were raised blocked the effect of the antibody. In contrast, affinity-purified W082, which did not recognize native proteins, did not alter regulation of phospholipase C. This indicates that the Gq family of signaling proteins can couple to several receptors and is responsible for the hormonal regulation of phospholipase C in these diverse systems. The further generality of this regulatory pathway remains to be established.     

Comparison of the effects of neurotensin and ACTH on the pituitary-adrenocortical axis of dexamethasone-suppressed rats.

Neurotensin (NT) (12-48 micrograms/kg-1/day-1, for 2 days, s.c.), like ACTH (60 micrograms/kg-1/day-1, for 2 days, s.c.), counteracted the dexamethasone (Dx)-induced (120 micrograms/kg-1/day-1, for 4 days, s.c.) adrenal zona-fasciculata cell atrophy. NT notably raised, in Dx-suppressed rats, the plasma concentration of ACTH, which reached about that found after exogenous ACTH administration. However, at variance with ACTH, NT did not enhance either plasma corticosterone (B) level or B production by adrenal quarters in vitro. The conclusion is drawn that NT modulates the function of the rat pituitary-adrenocortical axis, by simultaneously stimulating hypophyseal ACTH release and inhibiting steroidogenesis at the adrenal level.     

Satelliten-Telemetrie beim Wei?storch (Ciconia ciconia) auf dem Wegzug — eine Pilotstudie

Zusammenfassung (1) 1991 konnten erstmals 4 mit Kleinsendern ausgerüstete Weißstörche mit Hilfe der Satelliten-Telemetrie auf Teilstrecken ihres Wegzugs bis zu 46 Tage lang verfolgt werden. Die japanischen Sender betrugen nur etwa 2 % des Körpergewichts der Vögel; die Ortung erfolgte durch das ARGOS-System. Die Versuchsvögel zeigten völlig normales Zugverhalten. — (2) Drei der in Brandenburg und Sachsen-Anhalt markierten Vögel waren Ostzieher und konnten über Strecken von etwa 640–4700 km verfolgt werden, 1 Storch bis zur ägyptisch-sudanesischen Grenze. Ein Westzieher konnte rund 1400 km bis zu den Pyrenäen geortet werden. — (3) Die Vögel wanderten individuell recht verschieden. 2 zogen weitgehend kontinuierlich bis in den Sudan bzw. zu den Pyrenäen, die anderen legten längere Pausen ein. Die ermittelten Zugstrecken verliefen recht geradlinig; Richtungsänderungen erfolgten vor allem an der Donau, den Karpaten, am Mittelmeer und auf der Sinai-Halbinsel. Tagesetappen betrugen mindestens bis zu 370 km, in einem Fall in 21 Tagen durchschnittlich 224 km/Tag. Die Zuggeschwindigkeit lag in der Größenordnung von 30–90 km/h. — (4) Verbesserte Sender mit längerer Lebensdauer und mehreren Ortungen pro Tag dürften es bald ermöglichen, individuelle Wanderrouten von Weißstörchen und anderen Großvögeln praktisch lückenlos zu ermitteln. Begleitmannschaften werden zudem die Zug- und Rastökologie mit Sendern ausgerüsteter Vögel mit erfassen können. Damit dürfte der Vogelschutz auf dem Zug eine neue Dimension gewinnen.

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Satellite tracking of White Storks during the autumn migratory period — a pilot study

Summary (1) In 1991 parts of the routes of White Storks migrating in autumn could be recorded for the first time by satellite tracking. Four individuals could be followed for up to 46 days. Transmitter weight accounted for only about 2 % of body mass. Locations were obtained by the ARGOS system. Migratory behaviour of the experimental birds appeared to be absolutely normal. — (2) The birds were equipped with transmitters in eastern Germany. Three of them followed the eastern migration route and could be tracked from 640 up to 4700 km, the latter reaching the borders of Egypt and Sudan. A western migrant could be followed over a distance of about 1400 km towards the Pyrenees. — (3) Migration showed considerable individual variation. Whereas in two birds migration was largely continuous towards the Sudan and the Pyrenees, respectively, the other birds rested for longer periods. The tracked migration routes were fairly straight. Marked directional shifts occurred towards the Danube valley, at the Carpathian mountains, the Mediterranean and on the Sinai. Capacity per day was at least 370 km. One bird covered 224 km/day on average during a period of 21 days. Migration speed ranged in the magnitude of 30–90 km/h. — (4) Improved transmitters with increased lifetime giving several locations per day will presumably allow to record migration routes of White Storks and other large birds more completely in the near future. Escorts should then be able to closely analyse the ecology of migration and staging of their test birds. These possibilities may give a new dimension to bird conservation measures during migration.

     

Melatonin Increases Serotonin N-Acetyltransferase Activity and Decreases Dopamine Synthesis in Light-Exposed Chick Retina: In Vivo Evidence Supporting Melatonin-Dopamine Interaction in Retina

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin N-acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-N-acetyltryptamine), and not affected by two other purported melatonin antagonists, N-acetyltryptamine and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, or 5-methoxytryptamine. The melatonin-evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.     

The effect of extrathyroidal conversion inhibitor from food-deprived animals on iodothyronines deiodination by pituitary and cerebral cortical homogenates in vitro

The influence of an inhibitor of iodothyronines' extrathyroidal conversion on T4, T3 and rT3 deiodination by adult pig pituitary and cerebral cortical homogenates has been investigated. The homogenates were incubated with T4, T3 and rT3 in the presence of 5 mM dithiothreitol and evaporated diethyl ether extracts of sera obtained from fed and starved (1-14 days) rabbits. The extracts had no influence either on T4 to T3 or on T4 to rT3 conversion in cerebral cortex. Deiodination of rT3 to 3,3'-T2 in that tissue was significantly inhibited only by the extracts of sera obtained from 4 days starved rabbits. Inner-ring deiodination of both rT3 and T3 was not changed by the extracts got from short-term (1-4 days) fasted animals but was significantly reduced by the extracts from long-term (7-14 days) food-deprived subjects. Pituitary conversion of T4 to T3 was diminished by 35% in the presence of sera extracts gained from 1-9 days fasted rabbits and by about 50% on day 14 of fasting, but only the latter change was statistically significant. Short-term fasting inhibited T4 to rT3 conversion on days 2 and 4. Both deiodinations of rT3 and 5-deiodination of T3 were affected by extracts of sera collected during long-term fasting.     

Male sex behavior and testosterone concentrations in gilts administered testosterone propionate

Two gilts were administered testosterone propionate and their subsequent plasma testosterone concentrations and male sex behavior were recorded. These were compared to testosterone concentrations and male sex behavior in boars. Testosterone propionate (75 mg) was administered to the gilts every other day for 20 days (induction scheme) and every 10 days there-after (maintenance scheme). Concentrations of testosterone in plasma were elevated to concentrations detected in the boars during the induction scheme. During the maintenance scheme, concentrations of testosterone appeared to be lower than in boars. At 20, 30 and 40 days following the first injection, sniffing, nosing and mating song behaviors were exhibited by the testosterone treated gilts similar in frequency to the boars. Mounting behavior was first detected 30 days following the first testosterone propionate injection, and by day 40, the frequency of mounting was greater than observed in boars.     

Prostaglandins contribute to the vasodilation induced by nicotinic acid

The significance of endogenously formed prostaglandins in the vasodilation induced by nicotinic acid (NIC) was investigated. The forearm venous plasma level of radioimmunoassayed PGE (R-PGE) and the forearm blood flow (FBF) were measured in 13 healthy male volunteers at rest and during infusion of NIC. Each subject was subsequently re-studied after pretreatment with the PG synthesis inhibitor, naproxen. In the absence of naproxen, NIC infusion resulted in an almost four-fold rise in the release of R-PGE and a 60% increase in FBF. Pretreatment with naproxen did not affect the basal release of R-PGE or the basal FBF but inhibited both the release of R-PGE and the increase in FBF following NIC. The data support the hypothesis that the vasodilating effect of NIC is largely dependent upon an increased vascular formation of PG.