Characterization of elastase associated with granulomatous tissue remodeling

Elastases have been reported to be involved in various types of tissue injury. In this study we detected hydrolytic activities for [3H]-elastin and Suc-Ala-Ala-Ala-pNA (SLAPN) in hepatic granulomas which became elevated in parallel with enlargement of the granulomas and disappearance of aldehyde-fuchsin-stained filaments in the lesions of mice infected with Schistosoma mansoni. The elastase was partially purified by gel filtration followed by anion-exchange chromatography. This enzyme has a molecular weight of 20-25k and hydrolyzed denatured collagen (azocoll), Glu-Pro-Val-pNA, SLAPN, and [3H]-elastin. Optimal pH was 7-8.5. It is a serine proteinase and distinct in its inhibitor profile from murine peritoneal macrophage elastase, which has been reported by others. Digestion of elastic fibers in vessel walls and fine fibrils in newly developed granulomas by the granuloma elastase was histochemically identified with aldehyde-fuchsin stain. These results indicate that a serine proteinase functions as a major elastase in granulomatous tissue remodeling and may account for the disappearance of elastic fibers and other elements of the matrix in fully developed granulomas.     

Chromium content of normal and pathologically altered lung tissue

Flameless atomic absorption spectrophotometry was used to determine the chromium (Cr) content of samples taken at autopsy from the lungs, bronchi, and regional hilar lymph nodes of 50 randomly selected patients from urban and rural areas; these patients were not known to have been excessively exposed to Cr. On the average, the Cr concentration in the lungs of patients younger than 40 yr of age was approximately 2 μg/g; for patients over 40, the average Cr values were between 5 and 15 μg/g dry wt. The highest values were found in samples from the apex of the lung. The Cr concentration in lung and lymph nodes increased in proportion to age and the degree of anthracosis. Chromium content in the bronchial wall was very low in all cases. Chromium values up to ten times greater as compared to age-matched average values were found in scarred lung tissue, probably caused by a postinflammatory lymph vessel blockade. Slightly elevated Cr values were found in smokers' lungs. Chromium values in tissue from primary lung carcinomas (n=9) were lower than those in neighboring lung tissue. Based on the results of this study the amount of Cr of lung and bronchial tissue does not appear to be associated with the induction of bronchial carcinoma.     

Neutrophil elastase is associated with serglycin on its way to lysosomes in U937 cells

Mutations in the neutrophil elastase (NE) gene have been postulated to interfere with normal intracellular trafficking of NE as an AP3-interacting membrane integrated protein and to cause severe congenital or cyclic neutropenia in humans. Here, we show that in U937 promonocytes NE is synthesized as a predominantly soluble proenzyme and is completely secreted in the presence of phorbol esters similarly to serglycin. Using chemical cross-linking NE is shown to be associated with serglycin as 34 kDa proenzyme in the trans-Golgi region of these cells indicating that it is delivered to lysosomes associated with serglycin.     

Broadband spectral verification of optical clearing reversibility in lung tissue

The increase of tissue transparency through sequential optical immersion clearing treatments and treatment reversibility have high interest for clinical applications. To evaluate the clearing reversibility in a broad spectral range and the magnitude of the transparency created by a second treatment, the present study consisted on measuring the spectral collimated transmittance of lung tissues during a sequence of two treatments with electronic cigarette (e-cig) fluid, which was intercalated with an immersion in saline. The saline immersion clearly reverted the clearing effect in the lung tissue in the spectral range between 220 and 1000 nm. By a later application of a second treatment with the e-cig fluid, the magnitude of the optical clearing effect was observed to be about the double as the one observed in the first treatment, showing that the molecules of the optical clearing agent might have converted some bound water into mobile water during the first treatment.     

Rat lung decellularization using chemical detergents for lung tissue engineering

Although pulmonary diseases account for a large number of deaths in the world, most have no treatment other than transplantation. New therapeutic methods for lung treatment include lung tissue engineering and regenerative medicine. Lung decellularization has been used to produce an appropriate scaffold for recellularization and implantation. We investigated 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) with sodium dodecyl sulfate (SDS) and Triton X-100 detergents for effecting rat lung decellularization. We evaluated using conventional histology, immunofluorescence staining and SEM methods for removing nuclear material while leaving intact extracellular matrix proteins and three-dimensional architecture. We investigated different concentrations of CHAPS, SDS and Triton X-100 for different periods. We found that 2 mM CHAPS + 0/1% SDS for 48 h was the best among the treatments investigated. Our method can be used to produce an appropriate scaffold for recellularization by stem cells and for investigations ex vivo and in vivo.     

Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.     

Competitive neutrophil elastase inhibitory isoflavones from the roots of Flemingia philippinensis

Flemingia philippinensis has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (1–16), including the most potent and abundant prenyl isoflavones (1–9), from the F. philippinensis plant. These prenyl isoflavones (2, 3, 5, 7, and 9) competitively inhibited NE, with IC₅₀ values of 1.3–12.0 μM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound 3 had an IC₅₀ = 1.3 μM, k₃ = 0.04172 μM⁻¹ min⁻¹, k₄ = 0.0064 min⁻¹, and K_i^app = 0.1534 μM. The K_ik/K_iv ratios (18.5 ∼ 24.6) for compound 3 were consistent with typical competitive inhibitors. The prenyl functionality of isoflavones significantly affected inhibitory potencies and mechanistic behavior by shifting the competitive mode to a noncompetitive one. The remaining flavonoids (10–16) were confirmed as mixed type I inhibitors that preferred to bind free enzyme rather than the enzyme-substrate complex. Fluorescence quenching analyses indicated that the inhibitory potency (IC₅₀) closely followed the binding affinity (K_SV).     

The involvement of nitric oxide,nitric oxide synthase,neutrophil elastase,myeloperoxidase and proinflammatory cytokines in the acute lung injury caused by phorbol myristate acetate

Phorbol myristate acetate (PMA) causes acute lung injury (ALI). The present study was designed to elucidate the role of nitric oxide (NO), inducible NO synthase (iNOS), neutrophil elastase (NE) and other mediators in the ALI caused by PMA. In isolated rat’s lungs, PMA at various doses (1, 2 and 4 μg/g lung weight) was added into the lung perfusate. Vehicle group received dimethyl sulfoxide (the solvent for PMA) 100 μg/g. We measured the lung weight changes, pulmonary arterial pressure, capillary filtration coefficient, exhaled NO, protein concentration in bronchoalveolar lavage (PCBAL) and Evan blue dye leakage. Nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and myeloperoxidase (MPO) in lung perfusate were determined. Histopathological examination was performed. We detected the iNOS mRNA expression in lung tissue. PMA caused dose-dependent increases in variables for lung changes, and nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and MPO in lung perfusate. The pathology was characterized by alveolar hemorrhagic edema with inflammatory cell infiltration. Scanning electron microscopy revealed endothelial damage. PMA upregulated the expression of iNOS mRNA. Our results suggest that neutrophil activation by PMA causes release of NE, upregulation of iNOS and a series of inflammatory responses leading to endothelial damage and ALI.     

Normal lung tissue complication probability in MR-Linac and conventional radiotherapy

PurposeTo study normal lung tissue (NLT) complications in magnetic resonance (MR) image based linac and conventional radiotherapy (RT) techniques.Materials and MethodsThe Geant4 toolkit was used to simulate a 6 MV photon beam. A homogenous magnetic field of 1.5 Tesla (T) was applied in both perpendicular and parallel directions relative to the radiation beam.Analysis of the NLT complications was assessed according to the normal lung tissue complication probability (NTCP), the mean lung dose (MLD), and percentage of the lung volume receiving doses greater than 20 Gy (V₂₀), using a sample set of CT images generated from a commercially available 4D-XCAT digital phantom.ResultsThe results show that the MLD and V₂₀ were lower for MR-linac RT. The largest reduction of MLD and V₂₀ for MR-linac RT configurations were 5 Gy and 29.3%, respectively.ConclusionMR-linac RT may result in lower NLT complications when compared to conventional RT.     

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.     

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.     

Effects of mesenchyme on epithelial tissue architecture revealed by tissue recombination experiments between the submandibular gland and lung of embryonic mice

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E-cadherin, β-catenin and tight junctional proteins are localized at the cell–cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non-cavitated mass of cells where E-cadherin/β-catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11-day lung epithelium cultured with 13-day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E-cadherin/β-catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15-day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.     

Human neutrophil elastase (HNE) inhibitory polyprenylated acylphloroglucinols from the flowers of Hypericum ascyron

In the course of an investigation of human neutrophil elastase (HNE) associated with inflammation, the extract of the flower parts of Hypericum ascyron showed a significant influence to HNE. The responsible metabolites to HNE inhibition were found to be eight polyprenylated acylphloroglucinols, PPAPs (1–8) which showed IC₅₀ ranges between 2.4 and 19.9 μM. This is the first report to demonstrate that PPAP skeleton exhibits potent HNE inhibition. The compounds 1–3 were characterized and newly named as ascyronone E (IC₅₀ = 4.3 μM), ascyronone F (IC₅₀ = 19.9 μM), ascyronone G (IC₅₀ = 4.5 μM) based on 2D-NMR spectroscopic data. In the kinetic analysis of double reciprocal plots, all the compounds showed noncompetitive behaviors to HNE enzyme with the remaining of K_m and the increase of V_max. The binding affinity levels (K_SV) by using fluorescence were sufficient to be able to prove that PPAPs (1–8) had compliant interaction with inhibitory potencies.     

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture

Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Confocal microscopy of cells implanted into tissue blocks: Cell migration in long-term histocultures

Summary In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, then implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.     

急性臭氧暴露对大鼠肺部细胞遗传毒性的影响*

目的: 探讨不同浓度臭氧急性暴露对大鼠肺部细胞的遗传毒性的影响。方法: 36只wistar大鼠随机分为对照组(过滤空气暴露)、臭氧暴露组(0.12 ppm、0.5 ppm、1.0 ppm、2.0 ppm、4.0 ppm)共6组,每组6只。以不同浓度的臭氧对大鼠进行动态染毒4 h后,取肺组织并分离单细胞,采用酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHdG),利用彗星实验、微核试验和DNA-蛋白质交联实验进行DNA和染色体损伤分析。结果: 与对照组相比,肺组织中8-OHdG含量从臭氧暴露浓度为0.12 ppm起即显著增加,在0.5 ppm时达到最高值。随着臭氧暴露浓度升高,彗星拖尾率逐渐上升,且存在明显的剂量-效应关系;DNA-蛋白质交联率有先升高后下降的趋势,且在2.0 ppm时达到最大值;而肺部细胞微核率尽管呈现出上升趋势,但与对照组相比无显著性差异。结论: 急性臭氧暴露在较低浓度(0.12 ppm)时即可导致大鼠肺部细胞的DNA损伤;而在较高浓度(4 ppm)时却未见显著的染色体损伤。     

Isolation and partial sequence of a Kunitz-type elastase specific inhibitor from marama bean (Tylosema esculentum)

An isolation procedure utilizing ammonium sulfate fractionation and affinity chromatography was used to purify an elastase inhibitor present in large amounts in marama beans (Tylosema esculentum). The protein appeared to be heterogeneous due to carbohydrate differences, demonstrating two bands on SDS gels with molecular weights of 17.8?kDa and 20?kDa. Partial sequence, derived from mass spectrometry, indicated that the protein is a Kunitz-type inhibitor distinct from other known plant serine protease inhibitors. The marama bean inhibitor is specific for elastase, with very low K_i for both pancreatic and neutrophil elastase. The quantity of elastase inhibitor present in marama beans is many times greater than in soybean or any other bean or nut source reported to date. This raises the question of why a bean found in an arid corner of the Kalahari Desert would be so rich in a very potent elastase inhibitor.