Novel azapeptide inhibitors of cathepsins B and K. Structural background to increased specificity for cathepsin B

Abstract: We have designed and synthesized a new series of azapeptides which act as potential inhibitors of cathepsin B and/or cathepsin K. Their structures are based upon the inhibitory sites of natural cysteine protease inhibitors, cystatins. For the synthesized azapeptides, the equilibrium constants for dissociation of inhibitor–enzyme complex, K_i, were determined. Comparison of these values indicated that all of the azainhibitors act much stronger toward cathepsin B. Z‐Arg‐Leu‐His‐Agly‐Ile‐Val‐OMe ( 7 ) proved to be approximately 500 times more potent for cathepsin B than for cathepsin K. To be able to explain the obtained experimental values we used the molecular dynamics procedures to analyze the interactions between cathepsin B and compound 7 . We also determined the structure of the most potent and selective cathepsin B azainhibitor by means of NMR studies and theoretical calculations. In this report, we describe SAR studies of azapeptide inhibitors indicating the influence of the conformational flexibility of the examined compounds on inhibition of cathepsins B and K.     

正常人外周血淋巴细胞 IL—2受体免疫放射测定

采用~(125)I 标记小鼠抗人IL—2受体(P55)的抗Tac(CD_(25))单克隆抗体,成功地建立了人IL—2受体的免疫放射分析法,动态观察了人外周血淋巴细胞经PHA 刺激24、48和72h 后IL—2受体表达的时间曲线,通过Scatchard 作图分析表明~(125)I—抗Tac 单克隆抗体与PHA 活化的上述三个时间点的T 淋巴细胞的最大结合容量(B_(max))分别为43000位点/细胞、54000位点/细胞和61000位点/细胞。本研究为临床IL—2受体检测提供一种简便的免疫放射测定法。     

固本延衰方对亚急性衰老模型小鼠IL-2及其受体表达的影响

目的:探讨固本延衰方延缓亚急性衰老模型小鼠衰老的机制。方法:将60只小鼠随机分为4组,除正常对照组外均皮下注射D-半乳糖造成衰老模型,并给予不同剂量的固本延衰方治疗,分别用放免法及流式法测定脾淋巴细胞白细胞介素2及其受体水平。结果:衰老模型组IL-2及其受体水平明显低于正常对照组(P<0.05),固本延衰方各剂量组IL-2及其受体水平明显高于衰老模型组(P<0.05,P<0.01)。结论:提示固本延衰方通过提高衰老机体淋巴细胞IL-2及其受体表达水平,从而调节机体免疫功能延缓衰老。     

Orthotopic placement of the dunning R3327 AT-3 prostate tumor in the copenhagen X fischer rat

Orthotopic placement of in vitro propagated Dunning R3327 AT-3 tumor cells resulted in a greater percentage of tumor takes and a two-fold shift in the exponential growth curve compared to flank implantation. The orthotopic tumor appeared to disseminate preferentially to regional lymph nodes, rather than to the lungs which is characteristic of flank tumors. The results suggest an important role of stromal-epithelial interactions in the growth of this tumor. © 1995 Wiley-Liss, Inc.     

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.     

Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I_K,Ca) were investigated in ramified murine brain macrophages. In order to induce I_K,Ca the intracellular concentration of nominal free Ca²⁺ was adjusted to 1μM. The Ca²⁺-activated K⁺ current of brain macrophages did not show any voltage dependence at test potentials between –120 and +30mV. A tenfold change in extracellular K⁺ concentration shifted the reversal potential of I_K,Ca by 51mV. The bee venom toxin apamin applied at concentrations of up to 1μM did not affect I_K,Ca. Ca²⁺-activated K⁺ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I_K,Ca at concentrations between 1 and 50nM. Tetraethylammonium (TEA) blocked 8.0% of I_K,Ca at a concentration of 1mM, whereas 31.4% of current was blocked by 10mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2mM for their ability to block I_K,Ca. La³⁺ reduced I_K,Ca by 72.8%, whereas Cd²⁺ decreased I_K,Ca by 17.4%; in contrast, Ni²⁺ did not have any effect on I_K,Ca. Ba²⁺ applied at a concentration of 1mM reduced I_K,Ca voltage-dependently at hyperpolarizing potentials. Received: 17 January / Accepted: 5 May 1997     

Simultaneous Quantification of an Enantiomer and the Racemic Compound of Ibuprofen by X-ray Powder Diffractometry

Purpose. An X-ray powder diffractometric method was developed for the simultaneous quantification of the relative amounts of the racemic compound (±) of ibuprofen (I) and S(+)-ibuprofen (II), when they occur as a mixture. Methods. The X-ray powder diffraction patterns of I and II show pronounced differences. This formed the basis for the determination of the relative amounts of I and II when they occur as a mixture. X-ray lines with d-spacings of 14.41 and 4.37 Å were unique to I and II, respectively. Mixtures containing different proportions of I and II were prepared which also contained lithium fluoride (III) as an internal standard. Results. A linear relationship was obtained when the intensity ratio (intensity of the 4.37 Å line of II/intensity of the 2.01 Å line of III) was plotted as a function of the weight fraction of II in the mixture. Similar results were obtained in the case of I. Using these standard curves, the weight fractions of I and II in 'unknown' mixtures were determined. The experimentally determined analyte concentration ranged between 98 and 104% of the true value. The relative error in the analyses of individual samples was < 10%. The minimum detectable weight fraction of I in II and II in I were 0.032 (3.2% w/w) and 0.034 (3.4% w/w), respectively. The minimum quantifiable weight fractions were 0.136 for I and 0.112 for II. Since the X-ray diffraction patterns of S(+)-ibuprofen and R(–)-ibuprofen are identical, the conclusions drawn regarding mixtures of I and II will also hold true in the quantitative analyses of mixtures of I and R(–)-ibuprofen.     

应用力学因素检测膝关节功能的实验研究

目的研究应用力学因素检测膝关节功能方法的有效性.方法利用测力平台测量得到人体下肢在运动过程中的力学曲线,用数学方法确定力学曲线的最大值F和曲线特征值R.结果给出51例骨关节炎患者的分析数据,并给出30例正常人的结果作为对照.结论应用力学因素可以有效地检测膝关节功能状态.     

喜炎平注射液联合维生素K3治疗小儿秋季腹泻的疗效观察

目的观察喜炎平与维生素K3联合治疗小儿秋季腹泻的疗效.方法将89例秋季腹泻患儿随机分为2组.治疗组采用喜炎平与维生素K3联合治疗,对照组则采用利巴韦林治疗,疗程5d.结果治疗组的总有效率为93.3%,显著优于对照组的65.9%(X2=10.37,P＜0.01).结论喜炎平与维生素K3联合治疗小儿秋季腹泻值得进一步推广.     

Regulation of β-cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of β-cell mass and function, demonstrated both in vitro and in vivo . The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of β cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of β-cell mass and function by Akt.