Antiparallel beta-sheets in the crystal structure of the heptapeptide Met-Glu-His-Phe-Arg-Trp-Gly (ACTH 4-10)

The conformation of the molecules in ACTH 4-10 has been determined as part of a study of the conformations of the biologically active N-terminal fragments of the adrenocorticotropic hormone (ACTH). ACTH 4-10 crystallizes in two different superstructures. The substructure considered in the present work, is monoclinic, space group C2, a = 25.75(1) A, b = 27.78(1) A, c = 20.35(1) A, beta = 114.0(1) degrees, Z = 8 molecules ACTH 4-10 plus 22 weight per cent solvent. The crystals contain antiparallel beta-sheets, the orientations of the side groups are not found, because of disorder. The present crystal structure and those of other linear oligopeptides emphasize that antiparallel beta-sheets are energetically favourable. It is very unlikely, however, that the ACTH 4-10 crystals contain the molecules in their biologically active form.     

Possible mechanisms of the divergent effects of ACTH4--10 and its analog containing the D-isomer of phenylalanine on behavior

The effect of two oligopeptides--ACTG4-10 and hexopeptide met-glu-his-D-phen-lis-L-phen (Dphen-GP) on memorizing the situation and on orienting-investigating reaction was studied in albino rats by the method of elaboration of food-procuring habit in T-maze and by the method of "open field". It was shown that these peptides in a dose of 15 mcg/kg with certain periods of administration, have an opposite effect on maze learning but a similar effect on memorizing in the "open field". ACTG4-10 slightly increases motor activity in the "open field", whereas Dphen-GP decreases it considerably. It is suggested that ACTG4-10 improves the formation of trace processes independently of the sign of reinforcement, whereas Dphen-GP selectively enhances defensive reaction and memorizing, connected with negative reinforcement.     

Effect of ACTH1-24 and ACTH4-10 on distribution of 3H-noradrenaline in the brain of adrenalectomized rats.

Painful stimuli led to a decrease of the radioactive catecholamine pool in adrenalectomized rats. Intraventricular administration of both tritiated noradrenaline and ACTH produced a greater decrease of the labelled catecholamine pool than in the control adrenalectomized rats in 12 to 18 hr following injection. Blocking of monoamino-oxidase activity or biosynthesis by systemic administration of Pargyline or alpha-methyl-tyrosine did not prevent the effect of ACTH on brain catecholamines. It is concluded that ACTH exerts a direct influence on the brain catecholaminergic system and that this effect might be involved in ACTH dependent behavioural responses.     

Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).     

Effect of (L-Phe7) and (D-Phe7) ACTH 4-7 and an ACTH 4-7 analog with prolonged action on acetylcholinesterase activity in the rat brain

ACTH4-7 and its long-acting analog stimulate acetylcholinesterase activity of different areas of the rat brain. Based on the data concerning the effect of an amino acid mixture equivalent to ACTH4-7 and actinomycin D on acetylcholinesterase activity of the white substance of the large hemispheres it is inferred that the oligopeptide-induced increase in the enzyme activity is linked with the induction of the synthesis of new acetylcholinesterase molecules.     

The binding of Semax, ACTH 4-10 heptapeptide, to plasma membranes of the rat forebrain basal nuclei and its biodegradation

The binding characteristics of the peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) to plasma membranes of basal nuclei of the rat forebrain and the dynamics of its degradation during its incubation with these membranes were studied. Binding of the homogeneously labeled [G-3H]Semax was shown to be time-dependent, specific, and reversible. Specific binding of the heptapeptide depended on calcium ions and was characterized by the dissociation constant of the ligand-receptor complex Kd = 2.41 +/- 1.02 x 10(-9) M and by the concentration of binding sites Bmax = 33.5 +/- 7.9 x 10(-15) mol/mg of protein. A method of studying Semax biodegradation in the presence of plasma membranes of rat brain was developed. It is based on the use of the peptide homogeneously labeled with tritium and on an HPLC analysis with UV detection at 220 and 254 nm of the peptide fragments formed. The half-life of Semax in the presence of the plasma membranes was demonstrated to be longer than 1 h. Dipeptidylaminopeptidases are considered to be the main enzymes responsible for its biodegradation; they successively cleave Semax to the HFPGP pentapeptide and the PGP tripeptide. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

An ACTH 4-9 analog impairs selective attention in man

Male adults were tested in a dichotic listening task, providing electrophysiological measures of selective attention. Subjects were tested twice, 60 min after oral administration of either 40 mg of ACTH 4-9 analog, or placebo. Averaged auditory evoked potentials (AEPs) to tone pips when attended and when unattended, EEG spectra, heart rate and blood pressure, and behavioral performance were measured during task performance. ACTH 4-9 analog treatment impaired selective attention as indicated a) by a diminished difference between evoked potential waveforms to attended and to unattended tone pips, b) by an impaired behavioral signal detection performance. Furthermore, frontal EEG theta activity slowed down after ACTH 4-9 analog. With time on task, however, there was no decay, but an improvement of selective attention after peptide administration. Differences in attention could not be due to concurrent changes in general cortical and autonomic arousal as indicated by EEG alpha activity, blood pressure and heart rate. Since separate analyses of the AEPs revealed an increased processing of the unattended tone pips in the ACTH 4-9 analog sessions the impaired selective attention under ACTH 4-9 analog may be described as an inability to suppress processing of irrelevant or distracting stimuli.     

The open-field behavior of neuroticized rats]

A detailed study has been carried out of the behaviour in the open field of the lines of rats with different functional state of the nervous system after prolonged neurotization in dependence on the phase of circadian rhythm and terms after the end of the influence. Significant changes have been shown by the amplitude of circadian rhythms of emotionality and motor activity. It is established that the reaction of the rats lines to prolonged neuroticizing action depends on the phase of the circadian rhythm, the time after its termination and on genetically determined level of excitability of the nervous system.     

Individual typologic characteristics of the open-field behavior of rats

By means of ethograms record and analysis, connection has been studied between the properties of rats behaviour organization in the open field, determining the level of behaviour entropy in this test, and the speed of conditioned reflexes formation in the Skinner chamber. According to behaviour entropy level the rats are significantly divided into four groups; the lowest speed of conditioned reflexes formation in the Skinner's chamber is observed in the animals of the first (low entropy) group, the highest--in the fourth (high entropy) group. The obtained data are discussed according to Pavlov's concepts on the characteristics of the basic nervous processes, determining individual-typological characteristics of the higher nervous activity of the animal. Conclusion is made that division according to the level of behaviour entropy in the open field test may serve as a safe method of express-estimation of the animals abilities to conditioned habits formation.     

Effect of ACTH 4-10 on passive avoidance of rats lacking vasopressin (Brattleboro strain).

The hypothesis that the effects of ACTH 4-10 on avoidance are mediated via the release of endogenous vasopressin was investigated. To test this hypothesis, we observed the effect of ACTH 4-10 on the passive avoidance of Brattleboro rats with diabetes insipidus resulting from a total genetic deficiency of vasopressin (DI) and Brattleboro rats without diabetes insipidus (HE). Normal Long-Evans rats (LE) were also included for comparison purposes. The results did not support the hypothesis. ACTH 4-10 did influence the passive avoidance of DI rats; this should not have occurred if the release of endogenous vasopressin is necessary for ACTH 4-10 to influence avoidance.     

Naltrexone-sensitive behavioral actions of the ACTH 4-9 analog (Org 2766)

The effects of the ACTH 4-9 analog (Org 2766) and the COOH-terminal tripeptide of Org 2766 (Phe-D-Lys-Phe; PDLP) on retrieval of one-trial learning passive avoidance behavior were compared with those of beta-endorphin, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin, des-Tyr1-[Met5]-enkephalin and des-enkephalin-gamma-endorphin (DE gamma E). Amounts of intracerebroventricularly administered Org 2766, PDLP, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin and DE gamma E, which induced a comparable attenuation of passive avoidance behavior were determined. Pretreatment with the opiate antagonist naltrexone prevented the attenuating effect of these peptides on passive avoidance behavior except that of DE gamma E. The attenuating effect of Org 2766 and of [Met5]-enkephalin was reversed to facilitation of passive avoidance behavior in the presence of naltrexone. Subcutaneous treatment with Org 2766 and [D-Phe7]-ACTH 4-10 decreased electrical self-stimulation behavior elicited from the medial septal area. Naltrexone prevented the inhibitory effect of Org 2766 on this behavior, but not that of [D-Phe7]-ACTH 4-10. Although the attenuating effect of PDLP on passive avoidance behavior was not reduced by pretreatment with [Met5]-enkephalin- or beta-endorphin-antiserum, and PDLP induced neither analgesia nor excessive grooming, the data suggest that the inhibitory effect of Org 2766 and PDLP on passive avoidance behavior and electrical self-stimulation are mediated by endorphin systems in the brain.     

The MSH/ACTH(4-9) analog Org2766 counteracts isolation-induced enhanced social behavior via the amygdala.

MSH/ACTH-like peptides influence social behavior induced by isolation It has been previously demonstrated that changes in locomotor activity as a result of isolation can be counteracted by Org2766 via the amygdala. The present study investigates whether isolation-induced changes in social behavior can also be affected by this peptide via the amygdala. A fully automated observation system was applied for detailed registration and analysis of movements of group-housed and 7-day isolated rats in a social interaction test. Administration of the MSH/ACTH(4-9) analog into the central nucleus of the amygdala elicited decreased locomotion, approach, and avoidance behaviors after isolation as compared to placebo-treated controls. However, general activity and social interest of group-housed rats were not affected by the MSH/ACTH(4-9) fragment. It is hypothesized that the amygdala is a site of action for neuropeptides in modulating social behavior.     

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes

Summary. Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.     

Effect an ACTH analog on the processes of learning and memory in rats

On the basis of the idea of the important role of neurotransmitter systems in realization of neuropeptide effects, the participation was studied of the monoaminergic systems in the mechanisms of the ACTH analogue influence on the processes of learning and memory in control animals and animals with a changed functional state of the monoaminergic systems. In parallel the influence was studied of the ACTH analogue on the content of the endogenic monoamines in various brain structures of rats. It has been shown that administration of the ACTH analogue in a dose of 10 mcg affects the elaboration and preservation of conditioned reflexes (CRs) of passive avoidance, CRs of two-side avoidance and labyrinth CRs only in conditions of changed functional state of the monoaminergic systems. Amnesia, usually elicited by 5-oxytryptophane and disulfiram is prevented by administration of the ACTH analogue. Administration of the ACTH analogue is accompanied by the intensification of serotonine metabolism in the midbrain and medulla and by an increase of noradrenaline content in the hypothlamus.     

The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats

Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4-10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post-test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm.     

Effects of perinatal alcohol on sexual differentiation and open-field behavior in rats

Prenatal alcohol treatment prolonged the gestation period by 1 day in 5 out of 11 mothers and decreased the anogenital distance of pups of both sexes measured at birth. There were no effects on body weights of the pups at birth, total number of pups per litter, or sex ratio. The postnatally alcohol-treated males (pups nursed by alcohol-drinking mothers) had a significantly earlier preputial separation than animals treated with alcohol prenatally only or controls. There were no differences among the groups on the parameters of adult masculine sexual behavior, plasma testosterone, weights of sex accessory glands, or open-field behavior. Animals treated perinatally with alcohol showed a significant inhibition of penile reflexes. Repeated testing, however, abolished this effect.     

Effects of bemitil and benzimidazole on behavior of rats in open-field test

Under conditions of the open-field test, we demonstrated that bemitil and benzimidazole injected intraperitoneally into rats in doses of 50 to 150 mg/kg suppress horizontal and vertical (motor and research) activities, as well as decrease the frequencies of episodes of grooming, defecation, and urination. Possible mechanisms underlying modifications of behavioral phenomena triggered by the above agents are discussed. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 85–90, January–February, 2006