Human serial learning: enhancement with arecholine and choline impairment with scopolamine.

Arecholine (4 milligrams), a cholinergic agonist, and choline (10 grams), a precursor of acetylcholine, significantly enhanced serial learning in normal human subjects. The subjects received methscopolamine prior to both arecholine and placebo injections. Conversely, scopolamine (0.5 milligram), a cholinergic antagonist, impaired learning and this impairment was reversed by arecholine and choline and the impairment after scopolamine were inversely proportional to the subject's performance on placebo; that is, "poor" performers were more vulnerable to both the enhancing effect of cholinergic agonist and precursor and the impairment after cholinergic antagonist than "good" performers.

REM sleep induction by physostigmine infusion during sleep.

Physostigmine (an anticholinesterase agent that increases acetylcholine at the synapse), in a dose of 0.5 milligram, was given intravenously to seven normal human volunteers. When injected during rapid eye movement (REM) sleep, physostigmine woke the subjects, and when injected during non-REM sleep, it induced REM sleep. This result suggests that cholinergic mechanisms play a role in the induction of REM sleep and in modulating cortical arousal mechanisms.

Faster cholinergic REM sleep induction in euthymic patients with primary affective illness.

Arecoline, a cholinergic muscarinic receptor agonist, induced rapid eye movement sleep significantly more rapidly in patients with primary affective illness in remission than in normal control subjects matched for age and sex. These results, and others, suggest that patients with primary affective illness may have a supersensitive cholinergic system both when they are ill and when their symptoms are in clinical remission.

Methoscopolamine inhibition of sleep-related growth hormone secretion. Evidence for a cholinergic secretory mechanism.

We have examined the effects of cholinergic blockade with 0.5 mg methscopolamine bromide, intramuscularly, on sleep-related and insulin-induced growth hormone (GH) secretion. 17 normal young men were studied; 8 had sleep studies, and 12 (including 3 who also had sleep studies) had insulin tolerance tests (ITT) with 0.1 U/kg of regular insulin. After an adjustment night in the sleep laboratory, saline control night and methscopolamine night studies were done in random sequence; study procedures included electroencephalographic, electromyographic, and electrooculographic recordings, and blood sampling every 20 min for hormone radioimmunoassays. Prolactin levels were also measured during sleep. For methscopolamine night studies, the mean overall control GH level of 2.89+/-0.44 ng/ml and the mean peak control GH level of 11.09+/-3.11 ng/ml were dramatically reduced to 0.75+/-0.01 and 1.04+/-0.25 ng/ml, respectively (P<0.0001 and <0.001). Despite virtual absence of GH secretion during the night in every study subject, no measured sleep characteristic was affected by methscopolamine, including total slow-wave sleep (12.1+/-2.6% control vs. 10.3+/-2.5% drug, P>0.2). Sleep prolactin levels were not changed by methscopolamine. In contrast to the abolition of sleep-related GH secretion, administration of methscopolamine had only a marginal effect on the GH response to insulin hypoglycemia. None of nine time points differed significantly, as was also the case with peak levels, mean increments, and areas under the curves (P>0.2). Analysis of variance did, however, indicate that the lower GH concentrations achieved during ITT after methscopolamine (average 31.7% below control) were significantly different than control concentrations. We conclude that the burst of GH secretion which normally occurs after sleep onset is primed by a cholinergic mechanism which does not influence slow-wave sleep. Cholinergic mechanisms do not appear to play an important role in sleep-related prolactin secretion. The contrast between the complete suppression of sleep-related GH release and the relatively small inhibitory effect on ITT-induced GH secretion suggests that the neurotransmitter mechanisms, and presumably the pathways, which subserve sleep-related GH secretion in man may be different from those which mediate the GH response to pharmacologic stimuli such as insulin.

Antimicrobial peptides with unusual amino acid compositions and unusual structures.

Antimicrobial peptides, which constitute an important component of innate immunity in animal and plant kingdom, are ubiquitously distributed in nature. However, they differ widely in their sizes, sequences and structures. On the basis of their structure they have been broadly classified into three classes: a) linear peptides with propensity for amphiphilic alpha-helical structure, b) peptides with beta or alphabeta structure stabilized by different number of disulfide bridges and c) peptides with over-representation of certain amino acids or unusual structures. Although considerable amount of work has been done on peptides of all the three classes, recent reviews have emphasized on peptides belonging to the first two classes. The present review focuses on the peptides belonging to the third group. The antimicrobial peptides discussed in this article include aromatic amino acid-rich peptides, (Pro-Arg)-rich peptides, unusual defensins and defensin-like molecules, unusual antimicrobial peptides from amphibians, bacteriocins with unusual structure and anionic antimicrobial peptides.

Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity.

Species right across the evolutionary scale from insects to mammals use peptides as part of their host-defense system to counter microbial infection. The primary structures of a large number of these host-defense peptides have been determined. While there is no primary structure homology, the peptides are characterized by a preponderance of cationic and hydrophobic amino acids. The secondary structures of many of the host-defense peptides have been determined by a variety of techniques. The acyclic peptides tend to adopt helical conformation, especially in media of low dielectric constant, whereas peptides with more than one disulfide bridge adopt beta-structures. Detailed investigations have indicated that a majority of these host-defense peptides exert their action by permeabilizing microbial membranes. In this review, we discuss structural and charge requirements for the interaction of endogenous antimicrobial peptides and short peptides that have been derived from them, with membranes.

The effect of physostigmine on normal human sleep and dreaming.

Physostigmine, an anticholinesterase that increases the action of brain acetylcholine, induces rapid eye movement (REM) sleep in normal humans. In this study we show that man dreams during physostigmine-induced REM sleep. Seventeen normal volunteers were pretreated with methscopolamine and received one intravenous infusion per night of either placebo or physostigmine either ten or 35 minutes after sleep onset. Subjects were awakened at specific times after infusion and interviewed regarding any sleep mentation prior to awakening. Results indicated that dreaming occurred during physostigmine-induced REM periods but that physostigmine did not alter mentation during non-REM sleep. These dreams were similar to spontaneous REM sleep dreams in content, vividness, unusualness, and emotionality.

Development and use of pharmacological probes of the CNS in man: evidence of cholinergic abnormality in primary affective illness.

The role of cholinergic mechanisms in the regulation of CNS functions was studied in normal humans. The purpose was to develop in vivo pharmacological techniques to assess central neurotransmitter activity in normals and apply them in primary affective illness and other neuropsychiatric conditions. Central cholinergic stimulation by cholinomimetics induced rapid eye movement (REM) sleep, dreaming, cortical arousal, and accelerated the REM cycle. Effects on memory included enhancement of serial learning, short-term consolidation of low-imagery words, and retrieval of clustered information. Analgesia and reduction of the P100 component of visual EEG evoked responses were also noted after physostigmine. Using the induction of REM sleep by arecoline as a "marker" of central cholinergic functioning, it was possible to (i) demonstrate the phenomenon of pharmacological denervation supersensitivity in normal humans after pretreatment with scopolamine, and (ii) demonstrate a significantly faster REM induction by arecoline in two groups of patients with primary affective illness (remitted and depressed) compared to normals. Rapid REM induction was found, not only in remitted patients who were drug-free for 2 weeks, but in patients who had never received somatic therapy. Pretreatment with scopolamine on three consecutive mornings also induced sleep changes at night in normals similar to several sleep abnormalities reported in primary affective illness. This was confirmed by a multivariate discriminate analysis program, which had previously been shown to separate depressed patients, insomniacs, and normals. On the basis of these data, it is proposed that cholinergic abnormalities may be present in primary affective illness during both the ill and the well states.

Circadian variation in the time of "switch" of a patient with 48-hour manic-depressive cycles.

A 43-year-old patient with regularly occurring 48-hr manic depressive cycles was studied intensively for about 2 years. While she was hospitalized, she was rated for manic behavior on a 15-point scale every 2 hr. Using predefined criteria, we have systematically analyzed 173 switches into mania and 171 switches out of mania with respect to their time of occurrence during the 24-hr day and the influence of time of switch on the intensity and duration of mania. A significantly higher number of switches into mania occurred at night (12 midnight to 8 AM) with peak incidence between 4 AM and 6 AM. Switches out of mania peaked between 10 PM and 12 midnight and also between 6 AM and 8 AM. Night switches into mania were also associated with a significantly higher peak mania rating than morning (8 AM to 4 PM) switches and higher rate of mania increment than morning and evening (4 PM to 12 midnight) switches. This indicates that time of day or circadian factors may determine the frequency and intensity of the manic process in this patient.

Elevated sleep and waking heart rates in anxious depressions.

Autonomic functioning, as measured by baseline heart rates during daytime (DHR) and during sleep (SLHR), was examined in 28 patients with major depressive disorder (MDD), 13 patients with panic disorder (PD), 28 patients with MDD and coexisting PD (MDD + PD), and 19 controls. DHR and SLHR in patients with PD or MDD did not differ from normals. Only the MDD + PD group showed significantly higher heart rates than controls. This pattern of results suggests a combined effect of MDD and PD, providing psychophysiological support for clinical findings of poorer prognosis in this group.

A twin study of cholinergic REM induction.

Seven monozygotic twin pairs were found to display concordance for sensitivity to REM sleep induction by arecoline (intraclass correlation (ri) of 0.69, F = 5.35, p less than 0.02). The REM1-REM2 interval on the placebo night did not show significant concordance (ri = 0.05, F = 1.11, p = 0.44). Our previous study suggested that increased sensitivity to the muscarinic agonist arecoline, as measured by time to onset of the second REM period during sleep, may be an indicator of vulnerability to affective disorder. The present finding of twin concordance in response to arecoline suggests genetic variation in sensitivity of CNS muscarinic cholinergic receptors. Such increased sensitivity may play an etiologic role in affective disorder.

Cholinergic REM induction response: separation of anxiety and depression.

Five groups of subjects underwent EEG sleep recordings, arecoline rapid eye movement (REM) induction response testing, and Schedule for Affective Disorders and Schizophrenia (SADS) interview. Group I: 20 patients with primary major depressive disorder (MDD) (endogenous) without any coexisting anxiety disorder; Group II: 19 primary MDD (endogenous) patients with secondary panic, GAD, or phobic disorders; Group III: 18 patients with primary anxiety disorder without coexisting MDD; Group IV: 14 patients with primary anxiety plus secondary MDD; Group V: 26 normal controls. Modified Research Diagnostic Criteria (RDC) were used for diagnosis, based on the SADS interview. There was considerable overlap of SADS scaled scores between patient groups, which is consistent with a heterogeneous clinical presentation of depressive and anxiety states. REM latency was significantly shorter in patients with primary MDD (without anxiety) as compared with that in patients with primary anxiety (no MDD) and normals. Arecoline REM induction response time was significantly shorter in both primary affective groups (I and II) as compared with primary anxiety (no MDD) patients and normal controls. REM latency and arecoline REM induction time was not significantly different between the primary anxiety groups (III and IV) and normals. The study highlights the use of biological markers in differentiating between clinical syndromes confounded by mixed or overlapping phenomenology.

Free and bound choline blood levels after phosphatidylcholine.

In six normal subjects we investigated the effects of oral phosphatidylcholine (lecithin) on the concentrations of plasma choline, erythrocyte choline, and choline-containing lipids. Plasma choline levels rose 1 hr after treatment and remained elevated for 8 hr, with peaks at 3 and 4 hr after phosphatidylcholine. Erythrocyte choline levels also rose, although the rise was slightly delayed relative to plasma choline. There was no change in the plasma choline-containing lipid concentration. These results demonstrate that, in normal subjects, oral phosphatidylcholine indices prolonged rises in plasma and erythrocyte choline concentrations and is therefore useful when such effects are desired.

Cholinergic regulation of mood and REM sleep: potential model and marker of vulnerability to affective disorder.

To test the hypothesis that depression and REM sleep share common cholinergic mechanisms the authors administered arecoline 25 min after completion of the first REM period to 14 patients with remitted bipolar affective disorder, 15 normal controls, and 5 subjects with a personal or family history of affective disorder. The second REM period occurred significantly sooner in the remitted patients than in the normal controls. The patients also had a significantly higher density of eye movements during the first REM period and a higher percentage of REM sleep. The authors believe that increased cholinergic sensitivity and REM density may be biological markers of increased vulnerability to bipolar affective illness.

Interaction of bovine seminalplasmin with Escherichia coli RNA polymerase in the presence of rifampicin.

The interaction of bovine seminalplasmin and rifampicin with E. coli RNA polymerase was studied using fluorescence spectroscopy. Both seminalplasmin and rifampicin are known to be the inhibitors for the initiation of RNA synthesis in E. coli. Rifampicin quenced the intrinsic fluorescence of RNA polymerase and seminalplasmin when excited at 280 nm. However, excess of seminalplasmin reversed the quenching of RNA polymerase fluorescence by rifampicin. Upon addition of rifampicin to the seminalplasmin-RNA polymerase complex, no change in fluorescence spectrum was observed. It appeared that although rifampicin could form complexes with RNA polymerase and seminalplasmin alone, no binding domain was available for rifampicin in the RNA polymerase-seminalplasmin complex. These observations are discussed in the light of the 'initiation site' of E. coli RNA polymerase.

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity.

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.

Identification of a second membrane-active 13-residue peptide segment in the antimicrobial protein, bovine seminalplasmin.

Seminalplasmin (SPLN) is a 47-residue protein from bovine seminalplasma having broad-spectrum antibacterial activity. The protein has no hemolytic activity. SPLN interacts with lipid vesicles and its antibacterial activity appears to stem from its ability to permeabilize the bacterial plasma membrane. Analysis of SPLN's primary structure, with respect to its relative hydrophobicity and hydrophilicity, revealed a segment, PKLLETFLSKWIG, more hydrophobic than the rest of the protein. A synthetic peptide corresponding to this region had not only antibacterial activity but also hemolytic properties. Analysis of the SPLN sequence based on hydrophobic moment plots has revealed a second segment, SLSRYAKLANRLA, which could be membrane active. A synthetic peptide corresponding to this region shows only antibacterial activity with no hemolytic activity.

The antibacterial peptide seminal plasmin alters permeability of the inner membrane of E. coli.

Seminal plasmin (SPLN) a 47-residue peptide, isolated from bovine seminal plasma, exhibits antibacterial activity against Gram-positive and Gram-negative bacteria. Although SPLN strongly inhibits the transcription of various natural and synthetic templates by E. coli RNA polymerase in vitro, it also associates with model membranes of phosphatidylcholine and phosphatidic acid. We have undertaken experiments to ascertain whether SPLN permeabilizes the bacterial inner membrane and thereby exerts its antibacterial activity, as in the case of recently isolated antibacterial peptides from mammalian sources. Our results show that SPLN affects the permeability properties of the bacterial inner membrane which is reflected by increased uptake of ortho-nitrophenylgalactoside (ONPG), which can normally be translocated only by protein transporters. SPLN has also been shown to act on the outer membrane, since divalent cations inhibit antibacterial activity.

Seminalplasmin and caltrin are the same protein.

The sequence of seminalplasmin, a basic antimicrobial and transcription-inhibitory protein from bovine seminal plasma, has been determined using an automated sequenator. This sequence is slightly different from that reported earlier by Theil and Scheit [(1983) EMBO J. 2, 1159-1163] and identical with that of caltrin, a Ca2+-transport-inhibitory protein of bovine seminal plasma. Caltrin and seminalplasmin are, therefore, the same protein.

Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin.

The antimicrobial and hemolytic activities of the 13-residue peptide indolicidin (ILPWKWPWWPWRR-NH2), present in bovine neutrophils, and its analogs have been determined with a view to gaining insight into the structural roles of tryptophan and proline. Peptides where proline was replaced by alanine and tryptophan by phenylalanine showed antibacterial activities comparable to that of indolicidin. The peptides do not exhibit a strong propensity to occur in either helical or beta-sheet conformation. The peptides also do not appear to exert their activity by permeabilizing the bacterial plasma membrane unlike other endogenous antibacterial peptides. The presence of tryptophan appears to be essential for hemolytic activity as the phenylalanine analog does not exhibit any hemolytic activity.