In the present experiments, we characterized the action of human/rat corticotropin-releasing factor (h/rCRF) and acute stress (1 hr of immobilization) on hippocampus-dependent learning and on synaptic plasticity in the mouse hippocampus. We first showed that h/rCRF application and acute stress facilitated (primed) long-term potentiation of population spikes (PS-LTP) in the mouse hippocampus and enhanced context-dependent fear conditioning. Both the priming of PS-LTP and the improvement of context-dependent fear conditioning were prevented by the CRF receptor antagonist [Glu(11,16)]astressin. PS-LTP priming and improved learning were also reduced by the protein kinase C inhibitor bisindolylmaleimide I. Acute stress induced the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) 2 hr after the end of the stress session. The CaMKII inhibitor KN-62 antagonized the stress-mediated learning enhancement, however, with no effect on PS-LTP persistence. Thus, long-lasting increased neuronal excitability as reflected in PS-LTP priming appeared to be essential for the enhancement of learning in view of the observation that inhibition of PS-LTP priming was associated with impaired learning. Conversely, it was demonstrated that inhibition of CaMKII activity reduced contextual fear conditioning without affecting PS-LTP priming. This observation suggests that priming of PS-LTP and activation of CaMKII represent two essential mechanisms that may contribute independently to long-term memory.

Glucagon-like peptide-1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic p-cell. To characterize the side-chain groups required for interaction of glucagon-like peptide-1 with its receptor, we performed binding studies with alanine-substituted glucagon-like peptide-1 analogues on RINmSF insulinoma cells. The binding affinity and biological activity of glucagon-like peptide-1 have been found to be sensitive to alanine exchanges in the N-terminal positions 1, 4, 6 and the C-terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10-12, 14, 16-21 and 25-30 do not change receptor affinity. These findings could be exploited to design glucagon-like peptide-1 agonists and probably antagonists for further physiological studies.Glucagon-like peptide-1 is a 30-residue gastrointestinal hormone released from the enteroglucagon cells (L-cells) in the small intestine [l -31. The post-translational processing of proglucagon in the small intestine leads to the generation of glucagon-like peptide-1, corresponding to positions 78 -108 of the human proglucagon precursor [3, 41. In v i m , glucagon-like peptide-1 increases insulin secretion from the isolated rat [S] and pig pancreas [2, 61, and from isolated rat islets [7]. In man, glucagon-like peptide-1 is released into the circulation postprandially, and glucagon-like peptide-1 infusions stimulate insulin release [ 81. Therefore, glucagon-like peptide-1 plays an important role in the postprandial regulation of insulin secretion. Recent studies have shown that glucagon-like peptide-1 also has an anti-diabetogenic effect by reducing the isoglycaemic meal-related requirement for insulin in patients with non-insulin-dependent diabetes mellitus [9][10][11].Receptors for glucagon-like peptide-1 have been characterized in RINm5F cells [12-141, a Abbreviations. Fmoc, N-9-fluorenylmethoxycarbonyl; [HlAIglucagon-like peptide-1 , glucagon-like peptide-I , with an alanine exchange in position 1 for the naturally occurring histidine residue. All other glucagon-like peptide-1 analogues are named accordingly with the first letter giving the naturally occurring amino acid in the glucagon-like peptide-1 sequence and the number following the first letter giving the position of the exchanged amino acid.Note. This work is dedicated to Werner Creutzfeldt, Professor emeritus of Medicine, Georg-August University of Gottingen, Germany, on the occasion of his 70th birthday. the gene encoding the receptor has been localized in humans [17]. So far, little is known about the structural requirements for glucagon-like peptide-1 binding to its receptor. Binding studies with N-terminal and C-terminal glucagon-like peptide-1 fragments have shown that the C-terminal domains of the glucagon-like peptide-1 molecule are important for receptor binding. The N-terminal fragment glucagon-like peptide-1 (7 -2.5) did not show receptor binding, indicating that longer N-terminal fragments are needed for receptor recognition [ 1 31. The glucag...

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Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).

The biosynthesis of the lipop-eptide antibiotic surfactin was studied in whole cells of Bacillus subtilis ATCC 21332 which incorporate t4C-labeled precursor amino acids directly into the product. P4C]Acetate appeared in the fatty acid portion of surfactin and was also partially converted into leucine. An enzyme was isolated and partially purified from a cell-free extract of the bacillus which catalyzes ATP-Pi-exchange reactions which are mediated by the amino acid components of surfactin. This activation pattern is consistent with a peptide synthesizing multienzyme which activates its substrate amino acids simultaneously as reactive aminoacyl phosphates.

In view of the observation that corticotropin-releasing factor (CRF) affects several brain functions through at least two subtypes of G protein-dependent receptors and a binding protein (CRFBP), we have developed synthetic strategies to provide enhanced binding specificity. Human͞rat CRF (h͞rCRF) and the CRF-like peptide sauvagine (Svg), differing in their affinities to CRFBP by two orders of magnitude, were used to identify the residues determining binding to CRFBP. By amino acid exchanges, it was found that Ala 22 of h͞rCRF was responsible for this peptide's high affinity to CRFBP, whereas Glu 21 located in the equivalent position of Svg prevented high affinity binding to CRFBP. Accordingly, [Glu 22 ]h͞rCRF was not bound with high affinity to CRFBP in contrast to [Ala 21 ]Svg, which exhibited such high affinity. Furthermore, the affinity of both peptides to either CRF receptor (CRFR) subtype was not reduced by these replacements, and their subtype preference was not changed. Thus, exchange of Ala and Glu and vice versa in positions 22 and 21 of h͞rCRF and Svg, respectively, serves as a switch discriminating between CRFBP and CRFR. On the basis of this switch function, development of new specific CRF agonists and antagonists is expected to be facilitated. One application was the modification of the CRF antagonist astressin (Ast), whose employment in animal experiments is limited by its low solubility in cerebrospinal fluid. Introduction of Glu residues into Ast generated with [Glu 11,16 ]Ast an acidic astressin, which efficiently antagonized in vivo the CRFR1-dependent reduction of locomotion induced by ovine CRF without detectable binding to CRFBP.

The trichlorides of yttrium, lanthanum, and the lanthanides react with methyllithium in diethyl ether in the presence of tetramethylethylenediamine (tmed) to give the complexes [Li(tmed)]3[Ln(CH3)6] with Ln = Y, La, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. From the reaction of lutetium tri-tertbutoxide, tmed, and tert-butyllithium in pentane, [Li(tmed)z] [Lu(t-C,H,),] has been obtained. The new compounds have been characterized by elemental analyses and IR and NMR spectra. The structure of [Li(tmed)]3[H~(CH3)B] has been elucidated through complete X-ray analysis. The crystals are rhombohedral with a = 1280.7 (20) pm, a = 79.84 (13)O, space group R3c, 2 = 2, D(ca1cd) = 0.96 g cmW3, R = 0.050, and 1535 observed reflections. The potential of methylating &unsaturated aldehydes and ketones has been explored for [Li(tmed)13[Pr(CH3),] and [Li(tmed)13[sm (CH3),], and it was found that 1,2 methylation is favored over 1,4 addition.