Purification and characterization of catalase from goat (Capra capra) lung

Catalase plays a major role in the protection of tissues from toxic effects of H₂O₂ and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127±2 Å. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with K_i values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.     

肺癌患者与肺结核患者血清蛋白的电泳分析

采用SDS聚丙烯酰胺凝胶电泳银染色的方法,对58例肺癌患者和32例肺结核患者的血清蛋白进行比较分析,发现有五种特异蛋白,分别存在于A区、C区、F区和G区。肺癌组和肺结核组比较,存在极显著性差异(P<0.01),提示有早期诊断肺癌的价值,有可能成为临床诊断肺癌的一个重要参考指标。     

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

Molecular and cellular remodeling of HepG2 cells upon treatment with antitubercular drugs

Drug-induced liver injury (DILI) is an adverse outcome of the currently used tuberculosis treatment regimen, which results in patient noncompliance, poor treatment outcomes, and the emergence of drug-resistant tuberculosis. DILI is primarily caused by the toxicity of the drugs and their metabolites, which affect liver cells, biliary epithelial cells, and liver vasculature. However, the precise mechanism behind the cellular damage attributable to first-line antitubercular drugs (ATDs), as well as the effect of toxicity on the cell survival strategies, is yet to be elucidated. In the current study, HepG2 cells upon treatment with a high concentration of ATDs showed increased perforation within the cell, cuboidal shape, and membrane blebbing as compared with control/untreated cells. It was observed that ATD-induced toxicity in HepG2 cells leads to altered mitochondrial membrane permeability, which was depicted by the decreased fluorescence intensity of the MitoRed tracker dye at higher drug concentrations. In addition, high doses of ATDs caused cell damage through an increase in reactive oxygen species production in HepG2 cells and a simultaneous reduction in glutathione levels. Further, high dose of isoniazid (50–200 mM), pyrazinamide (50–200 mM), and rifampicin (20–100 µM) causes cell apoptosis and affects cell survival during toxic conditions by decreasing the expression of potent autophagy markers Atg5, Atg7, and LC3B. Thus, ATD-mediated toxicity contributes to the reduced ability of hepatocytes to tolerate cellular damage caused by altered mitochondrial membrane permeability, increased apoptosis, and decreased autophagy. These findings further emphasize the need to develop adjuvant therapies that can mitigate ATD-induced toxicity for the effective treatment of tuberculosis.     

Fluorescence Microscopy of Fresh Tissue as a Rapid Technique for Assessing Early Injury to Mesophyll

A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wavelengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.     

Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii^−/−) mice. The effects of arg-ii^−/− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii^−/− cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii^−/− mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.     

IL-33-ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL-33-ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL-33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL-33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL-33-ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL-33–ST2-AMs axis for the diagnosis and intervention of BPD.     

Inhibitory role of bone marrow mesenchymal stem cells-derived exosome in non-small-cell lung cancer: microRNA-30b-5p,EZH2 and PI3K/AKT pathway

Exosomal microRNA (miRNA) exerts potential roles in non-small-cell lung cancer (NSCLC). The current study elucidated the role of miR-30b-5p shuttled by bone marrow mesenchymal stem cells (BMSCs)-derived exosomes in treating NSCLC. Bioinformatics analysis was performed with NSCLC-related miRNA microarray GSE169587 and mRNA data GSE74706 obtained for collection of the differentially expressed miRNAs and mRNAs. The relationship between miR-30b-5p and EZH2 was predicted and confirmed. Exosomes were isolated from BMSCs and identified. BMSCs-derived exosomes overexpressing miR-30b-5p were used to establish subcutaneous tumorigenesis models to study the effects of miR-30b-5p, EZH2 and PI3K/AKT signalling pathway on tumour growth. A total of 86 BMSC-exo-miRNAs were differentially expressed in NSCLC. Bioinfomatics analysis found that BMSC-exo-miR-30b-5p could regulate NSCLC progression by targeting EZH2, which was verified by in vitro cell experiments. Besides, the target genes of miR-30b-5p were enriched in PI3K/AKT signalling pathway. Animal experiments validated that BMSC-exo-miR-30b-5p promoted NSCLC cell apoptosis and prevented tumorigenesis in nude mice via EZH2/PI3K/AKT axis. Collectively, the inhibitory role of BMSC-derived exosomes-loaded miR-30b-5p in NSCLC was achieved through blocking the EZH2/PI3K/AKT axis.