Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag? octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH₂ (peptide l), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH₂ (peptide 2), which closely corresponds to the original Flag? sequence. Enrichment of the peptides with ¹⁵N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

盘龙七片治疗不稳定性冠心病心绞痛临床观察

对５８例不稳定性冠心病心绞痛患者，用盘龙七片（３８例）及复方丹参片（２０例）治疗，观察治疗前后心电图ＳＴ段，血液流变学及临床症状的变化，结果：盘龙七片组心电图ＳＴ段的缺血变化明业优于复方丹参片组（Ｐ〈０．０１），血浆粘度及纤维蛋白原下降亦明显（Ｐ〈０．０５或Ｐ〈０．０１），结论：盘龙七片虽是一种抗风湿，骨伤的中成药，但同样对治疗不稳定性冠心病心绞痛有一定的疗效。     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice

The objective of this study was to determine whether the development of tolerance to CP 55,940, a potent cannabinoid agonist, was due to changes in the receptor or second messenger system. ICR mice treated with CP 55,940 (2 mg/kg) twice a day for 6 and one-half days developed a high degree of tolerance to the pharmacological effects of CP 55,940. The ability of CP 55,940 to produce motor hypoactivity, hypothermia and immobility was reduced 163-, 97- and 19-fold, respectively. Evaluation of 3H-CP 55,940 binding to rat brain membranes indicated no difference in receptor affinity between the vehicle- and CP 55,940-treated animals. However, these binding studies revealed a 50% decrease in receptor number in the cerebellum of the CP 55,940-tolerant mice. Although cAMP is generally considered to be the second messenger for cannabinoid receptors, little difference was observed in the inhibitory effects of CP 55,940 on adenylyl cyclase activity in cerebellum between vehicle and drug-treated mice. However, there was an increase in receptor mRNA which suggests a compensation for receptor loss. There are several possible explanations for these results. There may be sufficient spare receptors such that CP 55,940-tolerant mice are capable of producing a maximal effect on the second messenger system. On the other hand, one could conclude that cannabinoid receptor down-regulation does not account for the development of tolerance to all of the effects of CP 55,940 in mice.     

成人原发性肾病综合征血容量与肾素,醛固酮,心钠素的关系

探讨成人原发性肾病综合征(NS)血容量与肾素、醛固酮及心钠素的关系。方法 应用~(113)mInCl标记转铁蛋白稀释法测定血容量,放免法测定血浆激素水平。对水肿期NS28例、正常26例及其中NS缓解期随访18例进行检测。结果 (1)水肿期NS血容量与正常组无差别,血浆肾素活性(PRA)、血管紧张素Ⅱ(ATⅡ)、醛固酮(Ald)、心钠素(ANP)水平均较正常组高,白蛋白、各激素水平与血容量无显著相关;(2)缓解期与水肿期比较,总的血容量无差异,Ald、ANP显著降低,PRA、ATⅡ则无明显差异;(3)Ald与24小时尿排钠(UNaV)显著负相关。结论 Ald和ANP是NS钠排泄的主要调节因子。     

A novel cognition enhancer NS-105 modulates adenylate cyclase activity through metabotropic glutamate receptors in primary neuronal culture

The effect of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105), a novel cognition enhancer, on adenylate cyclase activity was investigated in cultured neurons of the mouse cerebral cortex. NS-105 (10^–7 and 10^–6 M) inhibited forskolin-stimulated cyclic AMP formation, an action that was dependent on pertussis toxin-sensitive G proteins. Conversely, in pertussis toxin-pretreated neurons, NS-105 (10^–7 –10^–5 M) significantly enhanced the forskolin-stimulated cyclic AMP formation, and this action was completely reversed by cholera toxin. A metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S, 3R-ACPD) produced similar bi-directional actions on the cyclic AMP formation. Both of these inhibitory and facilitatory actions of NS-105 and 1S, 3R-ACPD were blocked by L(+)-2-amino-3-phosphopropinoic acid (L-AP3). NS-105 (10^–6 M) and 1S, 3R-ACPD (10^–4 M) significantly enhanced isoproterenol- and adenosine-stimulated cyclic AMP formation. The enhancement of such Gs-coupled receptor agonists-stimulated cyclic AMP formation was also produced by quisqualate but not by L(+)-2-amino-4-phosphonobutanoate (L-AP4). The phosphoinositides hydrolysis was enhanced by 1S, 3R-ACPD (10^–4 M) but not by NS-105 (10^–6 M), however, 1S, 3R-ACPD-induced increase in phosphoinositides turnover was attenuated by NS-105. These findings suggest that NS-105 stimulates metabotropic glutamate receptor subclasses that are coupled both negatively and positively to adenylate cyclase, but it acts as an antagonist at the receptor subclasses that are linked to phosphoinositides hydrolysis. Received: 3 February 1997 / Accepted: 25 April 1997     

Human Y-79 retinoblastoma cells express functional corticotropin-releasing hormone receptors

In human Y-79 retinoblastoma cells corticotropin-releasing hormone (CRH) produces a marked and rapid increase of adenylate cyclase activity. The concentration of the peptide producing half-maximal stimulation is 60 nM. The effect of CRH is significantly antagonized by the specific CRH receptor antagonist alpha-helical CHR 9-41 and is mimicked by sauvagine and urotensin I, two peptides displaying sequence homology with CRH. These results demonstrate the presence of functional CRH receptors in human Y-79 retinoblastoma cells and suggest that this cell line may be a suitable model in which to study the action of CRH on human retinal cell function.     

The Recognition of Hypothalamo-Neurohypophysial Functions by Developing T Cells

Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.     

Solid-phase synthesis of oxytocin using iodotrichlorosilane as Boc deprotecting agent

Deprotection of the tert-butoxycarbonyl group during solid-phase synthesis of peptides can be conveniently and efficiently carried out using a neutral reagent, silicon tetrachloride/sodium iodide (iodotrichlorosilane). This simple and rapid method has been advantageously employed during the solid-phase synthesis of the pituitary hormone, oxytocin.