Derivatization of phosphopeptides with mercapto- and amino-functionalized conjugate groups by phosphate elimination and subsequent Michael addition.

Kinetics of the beta-elimination of the phosphate group from H-Tyr-Ser(PO3H2)-Phe-OH and H-Tyr-Thr(PO3H2)-Phe-OH and subsequent addition of thiols and amines to the dehydroalaninyl and beta-methyldehydroalaninyl residues formed, were followed by RP HPLC under alkaline conditions in the absence and presence of Ba2+ ions. By this reaction sequence, the phosphoserinyl peptide was conjugated with mono-N-(2-mercaptoethyl)amide of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (4), a mercapto-functionalized pentapeptide, H-His-Gly-Gly-His-Gly-NH(CH2)4SH, and an amino-functionalized fluorescent dye, 5-dimethylaminonaphthalene-1-[N-(5-aminopentyl)]sulfonamide (dansyl cadaverine). The beta-methyldehydroalanine residue was, in turn, observed to be a poor Michael acceptor.

Inhibitory effects of different galanin compounds and fragments on osmotically and histamine-induced enhanced vasopressin secretion in rats.

The effects of rat, porcine and human galanin, and the human 1-16 and human 16-30 terminal galanin fragments on vasopressin secretion were studied in rat. The plasma vasopressin level was determined by radioimmunoassay (RIA). There were no changes in the basal vasopressin secretion after galanin administration. A significant increase in vasopressin concentration was detected following 2.5% NaCl or histamine administration. I.c.v. injected rat, porcine or human galanin or the 1-16 N-terminal galanin fragment prevented the plasma vasopressin level enhancement. Following the i.v. administration of rat galanin or the i.c.v. injected 16-30 C-terminal galanin fragment, the vasopressin concentration did not return to the normal level. Administration of the galanin antagonist galantid (M15) i.c.v. before the rat galanin i.c.v. injection prevented the inhibitory effect on the increased plasma vasopressin level following 2.5% NaCl solution or histamine administration. The results indicate that there is no significant difference in the inhibitory effect of rat, porcine or human galanin or the 1-16 galanin fragment on the enhanced plasma vasopressin secretion induced by hyperosmosis or histamine administration. Our findings suggest that galanin, as a peptide modulator, is physiologically involved in the regulation of vasopressin release following different forms of stimulation: an osmotic response or histamine administration.

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.

Thermodynamics of 7-methylguanosine cation stacking with tryptophan upon mRNA 5' cap binding to translation factor eIF4E.

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5'-5' triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5' cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.

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 Alzheimer's 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.

Enantioselective direct aldol addition of acetone to aliphatic aldehydes.

The asymmetric direct aldol addition of acetone to aliphatic aldehydes catalyzed by D-proline, L-proline, and its derivatives was studied. While excellent results could be obtained in neat acetone using alpha-branched aldehydes, unbranched and beta-branched aldehydes gave moderate results. Two dipeptide derivatives, L-Pro-L-Try-CH(2)OH and L-Pro-L-Trp-OCH(3), were prepared and tested in this reaction and both were found to be able to induce enantioselectivities. The ee-values in the case of some aldehydes approached that obtained with L-proline. Immobilization of L-proline on a polystyrene resin by its carboxylic group provided a catalyst which is able to induce enantioselectivity, can be easily removed from the reaction mixture, and reused without a significant decrease in the enantioselectivity of the beta-hydroxyketones obtained in the cross-aldol additions.

Human amyloid-beta causes changes in the levels of endothelial protein kinase C and its alpha isoform in vitro.

Amyloid-beta (A(beta)) deposits and neurofibrillary pathology are characteristic features of Alzheimer's disease (AD). The association of A(beta) 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 Abeta toxicity and the altered PKC levels in cerebral endothelial cells. In this study, cultured brain endothelial cells exposed to A(beta)1-40 revealed a translocation of PKC from the membrane fraction to the cytosol. The content of the isoform PKC(alpha), 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 A(beta)1-40 treatment. These data suggest that the accumulation of A(beta) peptide in the cerebral vasculature may play a significant role in the down-regulation of PKC seen in the AD cerebral vasculature.

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.