Prolonged antinociceptive activity of pseudodipeptide analogues of Lys-Trp(Nps) and Trp(Nps)-Lys.

Peptide bond substitution in the molecules of Lys-Trp(Nps) (LTN) and Trp(Nps)-Lys (TNL) by an aminomethylene and ketomethylene bond, respectively, afforded pseudodipeptides with analgesic activity. The new compounds Lys psi(CH2NH)-Trp(Nps)-OMe (LTNAM) and Trp(Nps)psi(COCH2)(R,S)-Lys (TNLKM) induced a dose-dependent and naloxone-reversible analgesia following intracerebroventricular (ICV) administration to mice. The antinociceptive effects were longer lasting compared to those induced by the parent compounds. The pseudodipeptides protected Met-enkephalin degradation by rat striatal slices and, combined with an ineffective dose of the opioid peptide, induced analgesia. LTNAM and TNLKM were as potent as LTN to inhibit brain aminopeptidase in vitro and ex vivo. An increased resistance to proteolysis of the pseudodipeptides may explain their prolonged analgesic activity.     

Synthesis of [Trp(1-But)9]- and [Trp(2,5,7-But3)9] corticotropin-(1--19)-nonadecapeptideamide (author's transl)]

The title analogues of corticoptropin-(1--19)-nonadecapeptidamide in which the tryptophan residue in position 9 has been replaced by tert-butylated tryptophan derivatives, were prepared according to the methods of conventional peptide synthesis in solution. Both analogues showed remarkable steroidogenic activity as measured by Sayers test. The unexpectedly high biological potency is discussed.     

Crystal structures of HIV-1 Tat-derived nonapeptides Tat-(1-9) and Trp2-Tat-(1-9) bound to the active site of dipeptidyl-peptidase IV (CD26)

CD26 or dipeptidyl-peptidase IV (DPPIV) is engaged in immune functions by co-stimulatory effects on activation and proliferation of T lymphocytes, binding to adenosine deaminase, and regulation of various chemokines and cytokines. DPPIV peptidase activity is inhibited by both Tat protein from human immunodeficiency virus (HIV)-1 and its N-terminal nonapeptide Tat-(1-9) with amino acid sequence MDPVDPNIE, suggesting that DPPIV mediates immunosuppressive effects of Tat protein. The 2.0- and 3.15-A resolution crystal structures of the binary complex between human DPPIV and nonapeptide Tat-(1-9) and the ternary complex between the variant MWPVDPNIE, called Trp(2)-Tat-(1-9), and DPPIV bound to adenosine deaminase show that Tat-(1-9) and Trp(2)-Tat-(1-9) are located in the active site of DPPIV. The interaction pattern of DPPIV with Trp(2)-Tat-(1-9) is tighter than that with Tat-(1-9), in agreement with inhibition constants (K(i)) of 2 x 10(-6) and 250 x 10(-6) m, respectively. Both peptides cannot be cleaved by DPPIV because the binding pockets of the N-terminal 2 residues are interchanged compared with natural substrates: the N-terminal methionine occupies the hydrophobic S1 pocket of DPPIV that normally accounts for substrate specificity by binding the penultimate residue. Because the N-terminal sequence of the thromboxane A2 receptor resembles the Trp(2)-Tat-(1-9) peptide, a possible interaction with DPPIV is postulated.     

Naltrexone-sensitive behavioral actions of the ACTH 4-9 analog (Org 2766)

The effects of the ACTH 4-9 analog (Org 2766) and the COOH-terminal tripeptide of Org 2766 (Phe-D-Lys-Phe; PDLP) on retrieval of one-trial learning passive avoidance behavior were compared with those of beta-endorphin, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin, des-Tyr1-[Met5]-enkephalin and des-enkephalin-gamma-endorphin (DE gamma E). Amounts of intracerebroventricularly administered Org 2766, PDLP, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin and DE gamma E, which induced a comparable attenuation of passive avoidance behavior were determined. Pretreatment with the opiate antagonist naltrexone prevented the attenuating effect of these peptides on passive avoidance behavior except that of DE gamma E. The attenuating effect of Org 2766 and of [Met5]-enkephalin was reversed to facilitation of passive avoidance behavior in the presence of naltrexone. Subcutaneous treatment with Org 2766 and [D-Phe7]-ACTH 4-10 decreased electrical self-stimulation behavior elicited from the medial septal area. Naltrexone prevented the inhibitory effect of Org 2766 on this behavior, but not that of [D-Phe7]-ACTH 4-10. Although the attenuating effect of PDLP on passive avoidance behavior was not reduced by pretreatment with [Met5]-enkephalin- or beta-endorphin-antiserum, and PDLP induced neither analgesia nor excessive grooming, the data suggest that the inhibitory effect of Org 2766 and PDLP on passive avoidance behavior and electrical self-stimulation are mediated by endorphin systems in the brain.     

Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.     

Effects of ACTH(1-24) and ACTH/MSH(4-10) on isolation-induced distress vocalization in domestic chicks

The effects of centrally administered ACTH(1-24) and ACTH(4-10) on isolation-induced distress vocalizations (DVs) were assessed in the presence or absence of social cues (mirrored and plain environments). A dose-response analysis indicated that ACTH(1-24) at doses of 0.5 nM and above increased DVs relative to controls when the animals were tested in mirrored or social environments which reduce baseline levels of calling. This effect, however, was short-lived (approx. 15 min). When tested again 1 hr after injection, the treated animals did not differ from controls. ACTH/MSH(4-10) had no effect on vocalization when the animals were tested immediately after injection, but marginally increased calling when animals were tested an hour later. In addition to vocalization changes, ACTH(1-24) induced squatting when animals were isolated in the test boxes, and yawning, head shaking, wing flapping and preening when animals were reunited after testing. ACTH(1-24)-treated chicks also exhibited longer latencies to close their eyes when they were held in the cupped hands of the experimenter. Taken together, the results suggest that ACTH(1-24) induces a central state of arousal in chicks that resembles fear/anxiety.     

Centrally administered N-terminal fragments of ACTH (1-10, 4-10, 4-9) display convulsant properties in rabbits

Electroencephalographic and behavioral effects of the following ACTH fragments: 1-4, 4-9, 4-11, 1-10, 4-10, 1-13, 1-17 and 1-24 were studied in rabbits. Sequences 4-9, 1-10 and 4-10 displayed some epileptic properties, i.e., they induced epileptic seizures (only electrographic or also behavioral) or increased hippocampal spiking. The 4-9 sequence seemed to be the common sequence responsible for these proconvulsant effects. The possible involvement of the enkephalinergic system is discussed.     

9-Isoleucine]ACTH1-24, a competitive antagonist of ACTH1-24 induced cyclic AMP and corticosterone production.

Substitution of tryptophan⁹ in ACTH_1–24 by isoleucine results in complete loss of biological activity. A dose of 3.4 × 10^?5 M per ml fails to stimulate corticosterone and cyclic AMP production. This analogue inhibits cyclic AMP production and corticosterone production induced by ACTH_1–24 in isolated adrenal cortex cells. The I₅₀ values for corticosterone and cyclic AMP inhibition are 2.3 × 10^?6 M and 3.4 × 10^?6 M respectively.     

Intranasal administration of ACTH(1-24) stimulates catecholamine secretion.

Previously, we reported that intranasal (IN) ACTH(1-24) administration stimulates adrenocortical steroid secretion in normal subjects. To determine the efficiency of transmucosal absorption of ACTH into the adrenal medulla, we measured serum cortisol, aldosterone, epinephrine, norepinephrine and dopamine levels after IN vs. intravenous (IV) administration of 250 microg ACTH(1-24) in 7 healthy adult men (mean age 21.7 +/- 1.2 yr; range, 21 - 24 yr). Blood was collected at 0, 30, 60 and 120 min after administration of ACTH(1-24), and the levels of adrenocortical steroids and catecholamines were measured by specific RIA and HPLC methods, respectively. There were no side effects associated with IN or IV ACTH administration. Consistent with the previous study, serum cortisol and aldosterone increased after IN administration of ACTH(1-24), peaking 30 min after administration. Sixty minutes after IN and IV administration of ACTH, epinephrine levels increased by 41.9 +/- 13.1 % and 63.3 +/- 11.8 %, respectively, and remained elevated throughout the sampling period. Thirty minutes after IN or IV administration of ACTH(1-24), plasma norepinephrine levels increased by 55.9 +/- 13.4 % and 73.7 +/- 15.0 %, respectively, peaking 30 min after ACTH(1-24) administration, and decreasing to basal levels within 60 min. Plasma dopamine levels did not change after IN administration of ACTH(1-24). Adrenocortical steroid and catecholamine levels did not increase after IN administration of saline. These results demonstrate that IN administration of ACTH(1-24) not only stimulates adrenocortical steroids, but also epinephrine and norepinephrine.     

Effects of continuous alpha (1-24) ACTH infusion in the dog

Dogs chronically infused with alpha (1-24) ACTH for 2 weeks showed continuous elevations in plasma ACTH, cortisol, and progesterone levels. Haematologic changes included immediate increases in numbers of mature neutrophils and monocytes and reduced numbers of eosinophils and lymphocytes. Haematocrits were also reduced with ACTH infusion. Whereas serum potassium levels fell in association with ACTH, serum sodium was unchanged. Activities of two serum enzymes of probable liver origin, alkaline phosphatase and alanine aminotransferase, increased gradually with ACTH treatment. Histologic examination of liver tissue revealed prominent hepatocellular vacuolisation. The trophic action of ACTH infusion was manifested by an increased adrenal gland weight and an enhanced cortisol response to a bolus ACTH injection given 1 day after the infusion ceased. Long-term infusion of ACTH resulted in haematologic, biochemical and morphologic changes resembling those observed in dogs with spontaneous pituitary-dependent hyperadrenocorticism.     

1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea: A novel cyclophilin A allosteric activator

Cyclophilin A (CypA) plays an important role in many physiology processes and its overexpression has been involved in many diseases including immune disease, viral infection, neuro-degenerative disease, and cancer. However, the actual role of CypA in the diseases is still far from clear, and a complete understanding of CypA is necessary in order to direct more specific and effective therapeutic strategies. Based on the screening of our in-house library through the isomer-specific proteolysis method, we find a CypA activator (1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea), compound 1a, which can increase CypA’s PPIase activity and give allosteric behavior. The binding affinity of compound 1a to CypA has been confirmed by Fortebio’s Octet RED system and the increased phosphorylation of ERK in H446 cells is observed by treatment with both compound 1a and CsA. In order to further evaluate the binding mode between the activator and CypA, the allosteric binding site and allosteric mechanism of CypA are investigated by molecular dynamics (MD) simulations in combination with mutagenesis experiments. The results show that the allosteric binding site of CypA is 7 Å away from its catalytic site and is composed of Cys52, His70, His54, Lys151, Thr152 and Lys155. Compound 1a binds to the allosteric site of CypA, stabilizing the active conformation of catalytic residues, and finally promotes the catalytic efficiency of CypA. We believe our finding of the CypA allosteric activator will be used as an effective chemical tool for further studies of CypA mechanisms in diseases.     

Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.     

Neurochemical effects of the synthetic ACTH4-9-analog Hoe 427 (Ebiratide) in rat brain

The ACTH4-9-analog Hoe 427 systemically injected in a dose range from 0.01-10 micrograms/kg caused a fall in acetylcholine (ACh) content in different brain areas of the rat. This effect occurred 0.5 hour after a single administration and lasted up to 24 hours. The decrease in ACh content induced by Hoe 427 was more pronounced when the animals were pretreated with dexamethasone (over 7 days 1 mg/kg SC, daily). Coadministration of the choline uptake inhibitor hemicholinium-3 (HC-3) and Hoe 427 potentiated the decrease in ACh content induced by HC-3. In the same dose range Hoe 427 acutely evoked an increase of the activity of the enzyme choline acetyltransferase as well as an elevation of brain cyclic GMP content. These data indicate that Hoe 427 enhances ACh metabolism in rat brain after systemic administration.     

Suppression of food intake by a complement C3a agonist [Trp5]-oryzatensin(5-9)

[Trp5]-oryzatensin(5-9) (WPLPR), an agonist peptide for complement C3a receptor, has been designed based on the C-terminal region of ileum-contracting peptide oryzatensin derived from rice protein. We previously reported that WPLPR has anti-analgesic and anti-amnesic activities after central or oral administration. In this study, we found a novel function of WPLPR on food intake. WPLPR suppressed food intake after intracerebroventricular or intraperitoneal (i.p.) administration at a dose of 3-30 nmol/mouse or 30-300 mg/kg, respectively, in fasted mice. Orally administered WPLPR at a dose of 300 mg/kg also decreased food intake. WPLPR decreased gastric emptying after i.p. injection at a dose of 300 mg/kg. The anorexigenic activity of WPLPR was blocked by cyclooxygenase inhibitor or antagonist for prostaglandin (PG) E receptor EP4 subtype. These results suggest that WPLPR decreases food intake through PGE2 production followed by EP4 receptor activation.     

Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.