L. Balaspiri

Deletion analogues of transportan

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

On the mode of action of an oxytocin derivative (Z-Pro-D-Leu) on morphine-dependence in mice.

The dipeptide Z-Pro-D-Leu appears to attenuate the development of physical dependence on morphine in mice, as measured by the stereotyped jumping response after naloxone challenge. Injected subcutaneously, 50 μg Z-Pro-D-Leu was required to produce this effect, while a hundred times smaller amount injected into the cerebral ventricles was sufficient to produce the same effect. In morphine-naive mice, 50 μg of the dipeptide did not change the dose-response curve for morphine, measured by the antinociceptive effect of the opiate on a hot-plate test. Although Z-Pro-D-Leu per se did not induce antinociception in the doses tested (50, 100 and 200 μg), the larger doses potentiated the effect of morphine. As a possible mode of the central action, the effect of the dipeptide on the levels of noradrenaline (NA) dopamine (DA) and serotonin (5-HT) in the brain has been measured. Two hours after a single acute injection of Z-Pro-D-Leu (50 μg), the level of NA in the lower brain stem (pons + medulla) was significantly reduced. Daily administration of the dipeptide for 3 days resulted in decreases in the levels of NA and DA in the brain stem. In morphine-dependent mice, the 5-HT and DA levels in the brain stem were reduced 30 min after naloxone challenge. The data support the idea that Z-Pro-D-Leu inhibits the development of morphine-dependence by a mechanism of action in the central nervous system. This effect seems to be distinct from an antagonism between the dipeptide and morphine at the receptor level. It is likey, on the other hand, that the effect is related to an influence of the dipeptide on monoaminergic neurotransmission in the brain stem.

Human amyloid-β causes changes in the levels of endothelial protein kinase C and its α isoform in vitro

Abstract Amyloid-β (Aβ) deposits and neurofibrillary pathology are characteristic features of Alzheimer’s disease (AD). The association of Aβ with cerebral vessels is an intriguing feature of AD. While there is considerable evidence of altered activities of the major isoforms of protein kinase C (PKC) in the vasculature and neurons of AD brains, little is known about the relationship between the Aβ toxicity and the altered PKC levels in cerebral endothelial cells. In this study, cultured brain endothelial cells exposed to Aβ1–40 revealed a translocation of PKC from the membrane fraction to the cytosol. The content of the isoform PKCα, involved in the regulation of amyloid precursor protein (APP) secretion, was decreased in the membrane-bound fraction of rat endothelial cells and increased in the cytosol after Aβ1–40 treatment. These data suggest that the accumulation of Aβ peptide in the cerebral vasculature may play a significant role in the down-regulation of PKC seen in the AD cerebral vasculature.

IDENTIFICATION OF OXYTOCIN AND VASOPRESSIN FROM NEUROHYPOPHYSEAL CELL CULTURE

Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Effects of amyloid-beta on cholinergic and acetylcholinesterase-positive cells in cultured basal forebrain neurons of embryonic rat brain

The neurotoxic effects of amyloid-beta(1-42) and amyloid-beta(25-35) (A beta) on cholinergic and acetylcholinesterase-positive neurons were investigated in primary cultures derived from embryonic 18-day-old rat basal forebrain. After various time intervals, the cultures were treated with 1, 5, 10 or 20 microM A beta for different time periods. The cholinergic neurons and their axon terminals were revealed by vesicular acetylcholine transporter immunohistochemistry and the cholinoceptive cells by acetylcholinesterase histochemical staining. To assess the toxic effects of these A beta peptides on the cholinergic neurons, image analysis was applied for quantitative determination of the numbers of axon varicosities/terminals and cells. The results demonstrate that, following treatment with 1 or 5 microM A beta for 5, 10, 30, 60 or 120 min, no changes in vesicular acetylcholine transporter immunohistochemical staining were observed. However, after treatment for 30 min with 10 or 20 microM A beta, the number of stained axon varicosities was reduced, and treatment for 2 h they had disappeared. In contrast, vesicular acetylcholine transporter-positivity could be seen in some of the neuronal perikarya even after 3 days after treatment. The acetylcholinesterase staining was homogeneously distributed in the control neurons. After A beta treatment, the histochemical reaction end-product was detected in some of the neuronal perikarya or in the dendritic processes near to the soma. It is concluded that the neurotoxic effects of A beta appear more rapidly in the cholinergic axon terminals than in the cholinergic and acetylcholinesterase-positive neuronal perikarya.

Die Herstellung von für die Peptidsynthese geeigneten, optisch aktiven Pipecolinsäurederivaten

ZusammenfassungZur Darstellung der optisch aktivenl- undd-Formen der den im Pflanzenreich häufig vorkommenden „nicht-proteinogenen” Aminosäuren zuzuzählenden Pipecolinsäure werden eine neue Spaltungsmethode und in Verbindung damit sechs neue N-Carbobenzoxy-l- und-d-Pipecolinsäurederivate sowie vier „aktivierte Ester” der N-Carbobenzoxy-l- und-d-Pipecolinsäure, die N-o-Nitro-phenylsulfenyl- und N-t-Butyloxycarbonylabkömmlinge der beiden Antipoden, beschrieben. Diese Derivate werden zur Synthese biologisch aktiver Oligopeptide empfohlen. Es wurden auch die physikalischen Daten der reinenl- undd-Pipecolinsäure ermittelt.AbstractA new method for the resolution of pipecolic acid, a non-proteinogenic amino acid frequently occurring in plants, is described. Six new benzyloxycarbonyl derivatives, four activated esters of benzyloxycarbonyll- andd-pipecolic acid and the o-nitrophenylsulfenyl andt-butoxycarbonyl derivatives ofl- andd-pipecolic acid, useful for the synthesis of biologically active oligopeptides, were prepared. The physical constants of purel- andd-pipecolic acid are reported.

Dose-related effect of the oxytocin fragment (prolyl-leucyl-glycinamide) on α-MPT-induced catecholamine disappearance and serotonin level in rat brain

Graded doses of Pro-Leu-Gly-NH(2) (3.5 x 10(?12), 3.5 x 10(?11), 3.5 x 10(?10) or 3.5 x 10(?9) mol) were administered into the lateral cerebral ventricle of rats. The noradrenaline level of the dorsal hippocampus was increased 30 min after a dose of 3.5 x 10(?10) mol Pro-Leu-Gly-NH(2). The dopamine level was increased in the dorsal hippocampus and in the striatum. The serotonin level was increased in the hypothalamus, in the striatum and decreased in the dorsal hippocampus. The catecholamine disappearance following 350 mg/kg of ?-methyl-p-tyrosine indicated an accelerated dopamine disappearance in the striatum for each dose studied, while the hypothalamic noradrenaline disappearance was inhibited by a dose of 3.5 x 10(?11) mol of Pro-Leu-Gly-NH(2). The data indicate that Pro-Leu-Gly-NH(2) induces dose and region-dependent changes in the cerebral monoamine metabolism. The striatal dopamine and hypothalamic serotonin metabolism appeared to be the most sensitive for intraventricular Pro-Leu-Gly-NH(2).

Amyloid precursor protein carboxy-terminal fragments modulate G-proteins and adenylate cyclase activity in Alzheimer's disease brain.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimers disease (AD) and age-matched control brains was compared. 10 microM APP(639-648)-APP(657-676) (PEP1) causes a fivefold stimulation in the [35S]GTPgammaS-binding to control hippocampal G-proteins. APP(657-676) (PEP2) and APP(639-648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649-669) (PEP3) showed no regulatory activity in the [35S]GTPgammaS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [35S]GTPgammaS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

A novel gastrin-processing pathway in mammalian antrum

An antiserum, L221, has been developed that is specific for the C-terminal region of the N-terminal tridecapeptide (i.e., 1-13) fragment of the acid-stimulating hormone, G17. In contrast to N-terminal G17 antisera previously used to estimate 1-13 G17, L221 does not cross-react with other N-terminal gastrin fragments or with C-terminal extensions of G17. Using L221 in conjunction with conventional gastrin antisera, and reversed-phase HPLC, it has been possible to identify in addition to 1-13 G17 a further, formerly unrecognised gastrin fragment, 1-11 G17, in stomach extracts. The production of 1-13 G17, 1-11 G17 and other gastrin forms such as the biologically active hexapeptide G6 which is known to occur naturally cannot be explained by tryptic cleavage of progastrin. Instead, their biosynthesis could be explained by the actions of an enzyme with an endopeptidase 24.11-like specificity. In porcine antrum, unsulphated and sulphated G17 are present in similar amounts, but unsulphated 1-13 G17 was about twice as abundant as sulphate 1-13 G17. This is consistent with previous in vitro findings that endopeptidase 24.11 has a higher affinity for the Ala-11-Tyr-12 and Gly-13-Trp-14 bonds in unsulphated G17, than in sulphated G17. The results suggest a novel albeit minor, processing pathway for gastrin biosynthesis in pig antrum involving an enzyme resembling endopeptidase 24.11.

Antiamnesic effects of D-pipecolic acid and analogues of Pro-Leu-Gly-NH2 in rats

The antiamnesic effects of prolyl-leucyl-glycinamide (PLG) and analogues of this tripeptide were investigated in rats. Retrograde amnesia was induced by electroconvulsive shock treatment and the degree of amnesia was characterized by the attenuation of one-trial learning passive avoidance response. PLG resulted in dose-dependent attenuation of retrograde amnesia. Structural modifications included N-terminal protection, substitution of the C-terminal NH2 group, replacement of the N-terminal amino acid, and replacement of the second amino acid of the tripeptide. Some tripeptides, all of them containing D-pipecolic acid instead of the N-terminal proline, were more effective than PLG. Therefore, D-pipecolic acid, D-pipecolamide and their N-terminally protected analogues were also investigated, and were found to have powerful antiamnesic effects.