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.     

Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients

Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinson's disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.     

复方丹参注射液干扰素治疗乙肝后肝纤维化

目的探讨复方丹参注射液联合干扰素治疗慢性乙肝患者的疗效。方法110例慢性乙肝患者，按随机方法分成①对照组30例，应用普通保肝药物治疗，疗程6个月；②丹参组30例，应用复方丹参注射液（每ml含丹参、降香各1g）30ml加入10％葡萄糖溶液300ml中静脉注射1个月；③IFN组30例，应用IFN—α 3MU，隔日一次肌内注射，3个月；④联合组20例，应用复方丹参注射液30ml加10％葡萄糖溶液300ml静脉注射1个月，IFN-α 3MU，隔日一次肌内注射，3个月。丹参组，IFN组和联合组保肝药物治疗同对照组。四组病例在性别、年龄、病程，治疗前肝功能等方面均无统计学差异。治疗前检测肝功能，肝炎病毒标志，血清HA、IV—C、PCI—Ⅱ，部分病例进行肝穿病理检查。治疗开始后每月检测肝功能，3个月（治疗后）和6个月（随访时）时检测血清HA、IV—C、PCⅢ及乙肝病毒标志，治疗后1年行肝穿病理检查。结果治疗前四组患者血清HA、PCⅢ、IV—C水平无统计学差异；治疗后丹参组、IFN组、联合组血清HA、FCⅢ、IV—C水平较治疗前及对照组有不同程度的降低。结论复方丹参注射液联合IFN治疗可使血清HA、PCⅢ、IV—C有明显下降，肝组织病理改变明显改善，为目前有效的慢性乙肝治疗措施。     

Bile-pancreatic juice exclusion promotes Akt/NF-kB activation and chemokine production in ligation-induced acute pancreatitis

Using a unique surgical model (the donor rat model), we showed previously that duodenal replacement of bile-pancreatic juice, obtained fresh from a donor rat, ameliorates ligation-induced acute pancreatitis. We hypothesize that bile-pancreatic juice exclusion from gut exacerbates Akt/nuclear factor-kB (NF-kB) pathway activation and induces chemokine production in ligation-induced acute pancreatitis. We compared rats with bile-pancreatic duct ligation to those with duodenal bile-pancreatic juice replacement fresh from a donor rat beginning immediately before duct ligation. Sham control rats had ducts dissected but not ligated. Rats were killed 1 or 3 hours after operation (n=7/group). Akt activation (immunoblotting, immune-complex kinase assay, and ELISA), inhibitory protein I-kB (I-kB) activation (immunoblotting), and production of chemokines MCP-1 and RANTES (ELISA) were measured in pancreatic homogenates. NF-kB was quantitated in nuclear fractions using electrophoretic mobility shift assay. Duct ligation produced significant increases in pancreatic Akt, IkB, and NF-kB activation and production of MCP-1 and RANTES. Activation of the Akt/NF-kB pathway and increased MCP-1 and RANTES production in response to duct ligation were significantly reduced by bile-pancreatic juice replacement (ANOVA, P<0.05). Bile-pancreatic juice exclusion stimulates Akt/NF-kB pathway activation and increases chemokine production in ligation-induced acute pancreatitis. Presented at the annual meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 16, 2005 (poster).     

丹参酚酸B盐对内皮素诱导的人肝星状细胞收缩的抑制作用及机制研究

目的 以培养的人肝星状细胞（HSC）为研究对象，观察内皮素-1（ET-1）对HSC的收缩作用及丹参酚酸B盐（SA-B）对ET-1的抑制作用，并探讨SA B抑制HSC收缩的作用环节和可能作用机制。 方法 采用酶消化、Nycodenz密度梯度离心的方法分离人HSC，以胶原凝胶收缩法观察ET-1对于传代HSC的收缩作用，以及低、中、高3种剂量的SA B对其不同的干预作用；将ET-1、SA-B直接添加于传代HSC的无血清培养液中，以激光共聚焦显微镜技术观测HSC内钙离子浓度的变化。 结果 胶原凝胶收缩实验显示，ET-1可诱导HSC收缩（P<0.01）；3种剂量的SA-B可明显抑制ET-1诱导的HSC收缩（均P<0.01）；加入ET-1后，HSC形态学改变明显，细胞数量减少，但SA B对这些改变也有抑制。激光共聚焦显微镜下观察，ET-1可刺激细胞内钙离子短暂迅速增加，加入SA-B后，该作用被明显抑制。 结论 SA-B能显著抑制ET-1诱导的HSC收缩，其抑制收缩的机制与降低HSC内的钙离子浓度有关。SA-B的抑制HSC收缩作用可能是其抗肝纤维化及门脉高压的机制之一。     

慢性乙型肝炎肝郁脾虚证的logistic回归及判别分析

目的归纳慢性乙型肝炎肝郁脾虚证的辨证要点。方法对慢性乙型肝炎肝郁脾虚证、肝郁证、脾虚证的症状及舌、脉进行logistic回归及判别分析。结果肝郁脾虚证logistic回归提取的症状为腹胀、倦怠乏力、腹痛、食欲不振、紧脉、便溏、胁肋疼痛、胁肋不适；判别方程包含的症状则为腹痛、腹胀、倦怠乏力、便溏、黄苔。结论肝郁脾虚证的辨证要点包括胁肋疼痛或不适、腹胀、腹痛、乏力、食欲不振、便溏。利用logistic回归和判别分析归纳本证诊断要点是可行的。     

Immunogenicity and safety of a novel yeast Hansenula polymorpha-derived recombinant Hepatitis B candidate vaccine in healthy adolescents and adults aged 10–45 years

The aim was to determine whether the immunogenicity of an investigational hepatitis B vaccine (spHB) is at least as high as that of a licensed control vaccine, Engerix B^®, and to evaluate its safety before inclusion in new pediatric combination vaccines. Two randomized, controlled, blind-observer, Phase 3 trials were performed: one in Argentina (344 participants aged 10–15 years, 10 μg HBsAg/dose) and one in Uruguay (344 participants aged 16–45 years, 20 μg HBsAg/dose). Both vaccines were given in a 0, 1, 6 month schedule to all participants with a baseline anti-Hep B antibody titer <0.6 mIU/mL. Antibody titers were measured pre-dose 1, 1 month after dose 2, pre-dose 3, and 1 month after dose 3. Statistical non-inferiority analyses were performed on seroprotection rates (SP) post-dose 3 (% with anti-Hep B titers ≥10 mIU/mL; delta non-inferiority limit of −10%). In both studies, SP for the spHB vaccine was 100% and the spHB vaccine was non-inferior in terms of SP to the licensed control vaccine. GMTs post-dose 3 were approximately 1.8- and 4.1-fold higher for spHB in the 10–15 year and 16–45 year age groups, respectively. Reactogenicity was low for each vaccine, after each dose. This highly immunogenic hepatitis B candidate vaccine was selected for further investigation as a component of new pediatric combination vaccines.     

脑缺血后抑郁大鼠皮层及海马IP3表达及中药的干预作用

目的：从磷脂酰肌醇（PI）信号传导通路上重要调节因子IP3的角度，探讨脑缺血后抑郁模型磷脂酰肌醇（PI）信号传导通路的变化及中药颐脑解郁方的干预作用。方法：在脑缺血后抑郁模型上，采用AlphaScreen受体竞争机制，观察脑缺血后抑郁大鼠前额叶皮层、海马IP3含量的变化及中药颐脑解郁方的干预作用。结果：脑缺血后抑郁模型大鼠皮层及海马IP3含量明显高于正常组，模型组IP3的含量明显高于治疗组。结论：脑缺血后抑郁模型大鼠脑皮层及海马IR含量增加，中药颐脑解郁方能使之明显下调，提示脑缺血后抑郁IPl含量增加，颐脑解郁方的抗抑郁作用可能与调节受体后PI信号通路有关。