Elastase: a predictive marker of inflammation and/or infection

The present study was undertaken to estimate elastase in biological fluids and assess its usefulness as an indicator of inflammation/infection. Elastase was measured in seminal plasma, serum, urine, and cervical specimens using a specific substrate and was expressed in arbitrary units (AU). It was found to be stable over a period of 3 weeks. The intra- and interassay variation of elastase assay was between 2.3 to 6.8% and 7.3 to 9.9%, respectively. The assay was validated by comparing it with other methods that are available for the detection of infections. Sensitivity of the assay indicating inflammation/infection in these samples varied between 70.9 to 87.3%. The results obtained suggest that the presence of elastase in specimens may be used as a nonspecific indicator and could be used to screen inflammation/infection in a limited resource setting.     

Elastolytic activity in women with stress urinary incontinence and pelvic organ prolapse

AIMS: Weakening of pelvic supportive tissues is thought to be a contributing etiology in female pelvic floor disorders such as stress urinary incontinence and/or pelvic organ prolapse (SUI/POP). Since elastin modulates the mechanical properties of supportive tissues, we examined elastase activity in vaginal tissue from women with pelvic floor dysfunction compared to asymptomatic controls, by comparing overall elastase activity, human neutrophil elastase, cathepsin K, and alpha-1 antitrypsin (a serine protease inhibitor) mRNA and protein levels. METHODS: Full-thickness peri-urethral vaginal wall tissues were collected from age and menstrual-phase matched SUI/POP and control women at the time of pelvic surgery. Elastolytic activity in the homogenized tissue was determined by the generation of amino groups from succinylated elastin. To quantify mRNA levels of each protein, quantitative competitive-PCR and confirmatory Western blot analyses were performed on the samples for human neutrophil elastase, cathepsin K, and alpha-1 antitrypsin. RESULTS: The mean elastolytic activity in vaginal tissues from the SUI/POP group was similar to that in the control group. With respect to the proteolytic enzymes, neither human neutrophil elastase nor cathepsin K differed between the two groups. However, alpha-1 antitrypsin mRNA and protein levels were significantly decreased in tissues from affected women. CONCLUSIONS: A significant decrease in alpha-1 antitrypsin expression was seen in tissues from women with SUI/POP compared to controls. This data suggest that altered elastin metabolism may contribute to the connective tissue alterations observed in pelvic floor dysfunction. Future investigations are warranted to help define the role of elastin turnover in pelvic floor dysfunction.     

肺泡表面活性物质在金黄地鼠实验性慢性阻塞性肺病中的防治作用

目的探讨肺泡表面活性物质(PS)对慢性阻塞性肺病(COPD)的防治作用.方法通过给金黄地鼠吸入烟雾或气道内注入弹性蛋白酶两种方法,经1～3个月建立COPD动物模型,并应用外源性PS进行防治.对动物肺组织行光镜、电镜观察及显微微机图像分析进行形态定量.免疫组织化学法观察肺泡Ⅱ型上皮细胞表面活性物质蛋白A(SP-A)的表达情况.结果吸烟3个月及气道内注入弹性蛋白酶1和3个月,动物有明显的慢性支气管炎和肺气肿的病理变化.应用PS防治1个月,肺组织病变无明显改善;防治3个月后,肺组织损伤明显减轻,肺泡Ⅱ型上皮细胞的数量明显多于模型组,SP-A的表达比模型组增加(P＜0.01).结论经3个月的外源性PS干预,可减轻吸烟及弹性蛋白酶对肺组织的损伤,保护肺泡Ⅱ型上皮细胞的结构和分泌PS的功能,从而可有效减轻COPD的肺部病变.     

In the cut and thrust of apoptosis, serine proteases come of age

Proteolysis is central to the systematic cellular degradation that occurs during apoptosis. Predominantly, caspases have been studied in this regard. However, increasing evidence suggests that certain serine proteases may also play a significant role in apoptosis. Not only are these serine proteases involved in apoptosis signalling pathways independently, but they may also interact with more classical mediators of apoptosis such as the caspases or Bcl-2 family proteins. Isolation of apoptosis-associated serine proteases and the use of specific inhibitors have helped to shed light on potential pathways in which they are involved. Despite the recent developments in the field, knowledge regarding the role of serine proteases in apoptosis remains limited, but it is clear that investigations are gathering momentum and such studies may herald a new and exciting departure in apoptosis research.     

Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease

The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.     

男性不育症患者精浆弹性硬蛋白酶的临床分析（附96例报告）

目的 观察精浆弹性硬蛋白酶在男性不育症的变化，探讨精浆弹性硬蛋白酶值与CASA有关参数之间的关系。方法2005年11月～2006年5月至我院男科门诊就诊的96例男性不育症患者，年龄22～43岁，平均29．87岁：采用精浆弹性硬蛋白酶定量检测和DNA荧光染色精子动（静）态图像分析系统进行检测分析。结果完成的96例患者中，共有14例发现精浆弹性硬蛋白酶值异常，占14．58％。结论精浆弹性硬蛋白酶的检测是一项对判断精子生育力有价值的指标。     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB⁺-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P′₁ position and it has a wide specificity for the P₁ position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH₂). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P′₁ position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P₁ position. A synthesis was demonstrated with αAbu, Nle, Nva and Phg.     

Molecular and structural basis of steroid hormone binding and release from corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG), a non-inhibitory member of the serine proteinase inhibitor (serpin) super-family, is the high-affinity transport protein for glucocorticoids in vertebrate blood. Plasma CBG is a glycoprotein with 30% of its mass represented by N-linked oligosaccharide chains. Its well-characterized steroid-binding properties represent a “bench-mark data set” used extensively for in silico studies of protein–ligand interactions and drug design. Recent crystal structure analyses of intact rat CBG and cleaved human CBG have revealed the precise topography of the steroid-binding site, and shown that cortisol-bound CBG displays a typical stressed (S) serpin conformation with the reactive center loop (RCL) fully exposed from the central β-sheet A, while proteolytic cleavage of the RCL results in CBG adopting a relaxed (R) conformation with the cleaved RCL fully inserted within the protein core. These crystal structures have set the stage for mechanistic studies of CBG function which have so far shown that helix D plays a key role in coupling RCL movement and steroid-binding site integrity, and provided evidence for an allosteric mechanism that modulates steroid binding and release from CBG. These studies have also revealed how the irreversible release of steroids occurs after proteolysis and re-orientation of the RCL within the R conformation. This recent insight into the structure and function of CBG reveals how naturally occurring genetic CBG mutations affect steroid binding, and helps understand how proteolysis of CBG enhances the targeted delivery of biologically active steroids to their sites of action.     

Elastase inhibitors

Two applications claim novel 1,4-bisphenyl-1,4-dihydropyridine derivatives; 2-hydroxy and 2-aminodihydropyridines, respectively. The compounds are claimed to be useful as neutrophil elastase inhibitors and also in the treatment of both chronic obstructive pulmonary disease and myocardial infarction. These compounds represent yet another use of the widely employed 1,4-dihydropyridine privileged structure.     

应用龈沟液弹性蛋白酶评价正畸保持期牙周组织的改建状况

目的检测龈沟液中弹性蛋白酶（Elastase，EA）在正畸前、中、后以及保持各阶段的变化，探讨EA水平波动对保持期牙齿稳定性的提示意义。材料和方法以拔除四个双尖牙的病例为研究对象，从北京大学口腔医院正畸科门诊搜集分别处于正畸治疗前、治疗中关闭间隙、治疗结束、保持3—6个月、保持12个月五个阶段的病例，每组20例。以下切牙、拔牙间隙区牙齿为检测对象，提取龈沟液及记录临床检查指标，检测龈沟液中EA浓度。结果牙周临床检查指标（菌斑指数、牙龈出血指数）变化相似，在各阶段的变化无统计学意义。EA水平在治疗中关闭间隙阶段表现出明显的升高趋势，有统计学意义；治疗结束时EA水平仍高于治疗前；保持阶段呈缓慢下降的趋势，但保持一年后下切牙区EA水平仍高于治疗前。结论EA在正畸过程中及保持期的变化对牙齿的稳定状态具有提示作用。保持一年还不足以保证牙齿处于稳定状态。