THE PERMISSIVE ROLE OF HYPERTHERMIA IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA OR d-AMPHETAMINE1

Abstract— l -DOPA or d -amphetamine administration disaggregates brain polyribosomes in animals maintained in an environment warm enough (26°C) so that the drugs concurrently elevate their body temperatures to above 39°C. The production of equivalent hyperthermia (by keeping control rats at ambient temperatures of 40–44° C) does not cause similar disaggregation of brain polysomes. Hence, the role of hyperthermia in the drug-induced disaggregation is permissive.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

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.     

A new human colon carcinoma line

Supported by the “Tumorzentrum Heidelberg-Mannheim”.     

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.