Tissue-specific protective properties of lithium: comparison of rat kidney, erythrocytes and brain.

Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation.

Arginine-, D-arginine-vasopressin, and their inverso analogues in micellar and liposomic models of cell membrane: CD, NMR, and molecular dynamics studies.

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [D-Arg(8)]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides ß-turns: in the 2-5 and 3-6 fragments. The inverso-analogues also adopt ß-turns in the 3-6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr(2), Phe(3), and Arg(8), important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr(2) and Phe(3) in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide-membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic-zwitterionic liposomes as well as the anionic-zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.

Synthesis of lucifensin by native chemical ligation and characteristics of its isomer having different disulfide bridge pattern.

The antimicrobial 40-amino-acid-peptide lucifensin was synthesized by native chemical ligation (NCL) using N-acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid-phase synthesis is imcompatible. Two models of ligation were developed: [15+25] Ala-Cys and [19+21] His-Cys. The [19+21] His-Cys method gives lower yield because of the lower stability of 18-peptide-His-Nbz-CONH2 peptide, as suggested by density functional theory calculation. Acetamidomethyl-deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1-6], [C3-5], and [C2-4], which limits its ability to form α-helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one-pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium.

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae).

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH2 (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.

Bioportide: an emergent concept of bioactive cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Toxicity study of antimicrobial peptides from wild bee venom and their analogs toward mammalian normal and cancer cells.

Recently, we have isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. These peptides (melectin, lasioglossins, halictines and macropin) and their analogs display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity and low or moderate hemolytic activity. Here we describe cytotoxicity of the above-mentioned AMPs and some of their analogs toward two normal cell lines (human umbilical vein endothelial cells, HUVEC, and rat intestinal epithelial cells, IEC) and three cancer cell lines (HeLa S3, CRC SW 480 and CCRF-CEM T). HeLa S3 cells were the most sensitive ones (concentration causing 50% cell death in the case of the most toxic analogs was 2.5-10 µM) followed by CEM cells. For the other cell lines to be killed, the concentrations had to be four to twenty times higher. These results bring promising outlooks of finding medically applicable drugs on the basis of AMPs. Experiments using fluorescently labeled lasioglossin III (Fl-VNWKKILGKIIKVVK-NH(2)) as a tracer confirmed that the peptides entered the mammalian cells in higher quantities only after they reached the toxic concentration. After entering the cells, their concentration was the highest in the vicinity of the nucleus, in the nucleolus and in granules which were situated at very similar places as mitochondria. Experiments performed using cells with tetramethylrhodamine labeled mitochondria showed that mitochondria were fragmented and lost their membrane potential in parallel with the entrance of the peptides into the cell and the disturbance of the cell membrane.

Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae).

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.

Lucifensin, a novel insect defensin of medicinal maggots: synthesis and structural study.

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.

Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus.

BACKGROUND: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule. RESULTS: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established. CONCLUSIONS: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

Study of oostatic peptide uptake and metabolism in developing ovaries of the flesh fly, Neobellieria bullata.

The uptake and metabolism of the oostatic pentapeptide analogue of trypsin modulating oostatic factor (TMOF), H-Tyr-Asp-Pro-Ala-Pro-OH (5P), in ovaries of Neobellieria bullata (Parker) (Diptera: Sarcophagidae) were analyzed during their developmental stages. During selected stages of yolk deposition, the fate of [3HPro(3)]5P after its in vivo injection was compared to its uptake after in vitro incubation of dissected ovaries. The ovaries were analyzed from 30 s to 180 min after incubation. A detection sensitivity of 60-100 fmol of the labeled 5P was achieved using radio-high performance liquid chromatography. While the uptake of the applied radioactivity strongly depended on the stage of vitellogenesis, especially for the in vitro experiment, degradation of 5P was very quick and independent of whether the label was injected or incubated with the ovaries, regardless of the developmental stage of ovaries. No tracers of 5P were detected at 30 s after applying the labeled 5P in all tests.

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors.

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Galpha(q) protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.

Lasioglossins: three novel antimicrobial peptides from the venom of the eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae).

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs.

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides possess strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5-40 microM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt alpha-helical conformation in such anisotropic environments.

Structure-activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

Cocaine- and amphetamine-regulated transcript (CART) peptide specific binding in pheochromocytoma cells PC12.

CART (cocaine- and amphetamine-regulated transcript) peptides have been studied for ten years. We report specific binding of 125I-CART(61-102) to the rat adrenal pheochromocytoma PC12 cell line, both intact cells and cell membranes. Saturation binding to intact plated cells resulted in Kd of 0.48+/-0.16 nM and Bmax of 2228+/-529 binding sites/cell. 125I-CART(61-102) was also bound to PC12 cells differentiated using nerve growth factor to the neuronal phenotype with non-specific binding below 20%, and Kd of 1.90+/-0.27 nM and Bmax of 11,194+/-261 binding sites/cell. In competitive binding experiments, CART(61-102), CART(55-102) and di-iodinated CART(61-102) were bound to PC12 cell membranes with Ki in low nM range; their affinity to intact non-differentiated and differentiated cells was in low 10(-8) M range. In order to prove that iodination did not eliminate the pharmacological properties of CART, we tested the biological activity of di-iodinated CART(61-102). It decreased food intake in in vivo feeding experiment on fasted mice in a dose of 1 microg/mouse to the same extent as CART(61-102) in a dose of 0.5 microg/mouse.

New bradykinin analogues acylated on the N-terminus: effect on rat uterus and blood pressure.

Our previous studies suggested that acylation of the N-terminus of several known B2 antagonists with various kinds of bulky acyl groups consistently improved their antagonistic potency in rat blood pressure assay. On the other hand, our earlier observations also seemed to suggest that the effects of acylation on the contractility of isolated rat uterus depended substantially on the chemical character of the acyl group, as we observed that this modification might either change the range of antagonism or even transform it into agonism. Bearing all this in mind, we decided to synthesize seven new analogues of bradykinin by N-terminal acylation with various acyl groups of a moderately potent B2 antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. The analogues were tested in vitro for their blood pressure-lowering and uterotonic activities. The modifications either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, all seven substituents negatively influenced the interaction with the rat uterine receptors. Our results may be helpful for designing new B2 agonists and antagonists.

Analogs of arginine vasopressin modified in the N-terminal part of the molecule with a conformationally constrained cis-peptide bond motif.

The present work is part of our studies aimed at clarifying the influence of steric constraints in the N-terminal part of arginine vasopressin (AVP) and its analogs on the pharmacological activity of the resulting peptides. We describe the synthesis of eight new analogs of AVP or [3-mercaptopropionic acid (Mpa)]AVP (dAVP) substituted at positions 2 and 3 or 3 and 4 with two diastereomers of 4-aminopyroglutamic acid. The steric constraints provided by this modification turned out, however, so strong that all the peptides were inactive in all of the bioassays (pressor, antidiuretic and uterotonic tests).

Effect of humanin analogues on experimentally induced impairment of spatial memory in rats.

Humanin and its analogues have been shown to protect cells against death induced by various Alzheimer's disease genes and amyloid-beta-peptides in vitro: the analogue [Gly14]-humanin has also been shown to be potent in reversing learning and memory impairment induced by scopolamine in mice in vivo. It is important to validate these results by using other behavioral methods. In this study, the effect of [Gly14]-humanin and des-Leu-PAGA, another analogue (0.2 micromol kg(-1), i.p.) on the 3-quinuclidinyl benzilate-induced (2 mg kg(-1), i.p.) impairment of spatial memory in the multiple T-maze in rats has been evaluated. Both peptides reversed the impairment of spatial memory. These results indicate the potential of humanin analogues in modulation of the cholinergic system.