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71.
Daniela Pinzani Anna Maria Papini Mario Chelli Mauro Ginanneschi Carlo Alberto Maggi Riccardo Patacchini Laura Quartara Gianfranco Rapi 《Letters in Peptide Science》1996,2(5):307-313
Summary The adamantane moiety was introduced in the tachykinin NK2 receptor-selective agonist [-Ala8]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH2, MEN 10210) and in different positions of the NK2 receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) in order to investigate how this substitution affects their biological activity at tachykinin NK1, NK2 and NK3 receptors. 1-Adamantaneacetic acid (1-Ada-CH2COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH2CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH2). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH2CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH2CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH2CO)-NH2]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK1 and NK2 receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK3 receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK1, NK2 and NK3 receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK2 receptors of the rabbit isolated pulmonay artery. 相似文献
72.
本实验采用酶联免疫分析法检测猪主动脉内皮细胞培养液中tPA抗原、tPA活性和PAI活性,并观察了LDL和L精氨酸对tPA和PAI的影响。结果表明:LDL能明显降低tPA抗原含量。同时伴有tPA活性的降低和PAI活性增高;L精氨酸能增加tPA抗原含量伴tPA活性增高,但对PAI活性无明显影响。实验提示L精氨酸能保护血管内皮细胞,增加其tPA合成,并能部分地拮抗LDL降低tPA合成的作用,可能对动脉粥样硬化早期防治有重要应用价值。 相似文献
73.
利用离休孵育脑薄片和放射免疫测定其释放的精氨酸加压素(AVP)方法,探讨糖皮质激素(GC)在不能进入细胞内的情况下,对去肾上腺大鼠的下丘脑薄片释放AVP的快速影响及其可能的细胞膜机制。结果如下:(1)下丘脑薄片能够稳定地释放AVP(2h),其释放量为15.42±1.28pg/min;(2)牛血清白蛋白耦联皮质酮(B-BSA)对AVP的释放具有快速的(20min)抑制性效应,在10 ̄(-7)─10 ̄(-4)mol/L范围内呈剂量一效应关系;(3)GC细胞内受体拮抗剂RU486(10 ̄(-4)─10 ̄(-3)mol/L)能部分地阻断B─BSA的快速抑制效应;(4)孵育液中Ca ̄(2+)程度升高,B─BSA的快速抑制效应明显增强;反之,孵育液中无Ca ̄(2+)则B-BSA的快速抑制效应有所减弱。表明GC在未进入细胞内的情况下也可快速地抑制大鼠下丘脑薄片释放AVP,因此没有通过传统的基因组机制,而是由非基因组机制介导的,其作用部位在细胞膜水平上,可能是影响Ca ̄(2+)的跨细胞膜内流通量或/和影响有Ca ̄(2+)参与的AVP释放过程的结果。 相似文献
74.
Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies 总被引:13,自引:0,他引:13
Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity
from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding
how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In
this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress
90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of
novel developments that have enhanced refolding yields. 相似文献
75.
76.
Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca2+-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca2+. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca2+ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca2+/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca2+/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca2+-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins. 相似文献
77.
Jean L. Sarcich H. David Fischer Merrill S. Babcock Joseph W. Leone Alfredo G. Tomasselli 《Journal of Protein Chemistry》1995,14(2):73-80
Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM
r
, ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP. 相似文献
78.
Maurizio Pellecchia Gerhard Wider Hideo Iwai Kurt Wüthrich 《Journal of biomolecular NMR》1997,10(2):193-197
A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain 1H and 15N resonances inuniformly 15N-labeled proteins. Correlations between1H, 1Hand 1H are established on the basis of3J(15N,1H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with 1H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly15N-labeled proteins of 7 and 23 kDa molecular weight. 相似文献
79.
J. H. Hurley A. C. Newton P. J. Parker P. M. Blumberg Y. Nishizuka 《Protein science : a publication of the Protein Society》1997,6(2):477-480
C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not. 相似文献
80.
Barbara Lubec Zhuang Ya-hua Sainio Pertti Tuohimaa Pentti Erwin Kitzmüller Gert Lubec 《Life sciences》1997,60(26):618-2381
L-arginine and taurine are still in the center of physiological and pharmacological research. Although the fate of nitrogen of both compounds and of the 35S-taurine is well-documented, the fate of the carbon skeleton has not been elucidated yet. We studied the organ distribution of 14C arginine and 14C taurine over time in the mouse using whole body autoradiography with densitometric image analysis. We describe different organ distribution patterns. Kidney, heart, lung, the Harderian gland, the central nervous system, intestine and testis showed a comparable pattern of arginine disappearance in contrast to rapid disappearance in the salivary gland and the accumulation pattern in bone and spleen. Data on 14C taurine of liver, kidneys, lung, testis and Harderian gland resembled the arginine pattern; Accumulation of taurine carbon was found in salivary gland, bone, intestine, heart and brain. Our studies challenge and demand further related studies to obtaining more information on the fate of the carbon skeleton of these amino acids. 相似文献