排序方式: 共有49条查询结果,搜索用时 93 毫秒
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Goutham VH Kamalesh MD Guruprasad PK Vadhiraja MB Satyamoorthy K Rao Bola Satish S 《Analytical biochemistry》2011,(2):287-293
Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose–response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose–response relationship for DSBs was observed at a cell number of 4 × 105 cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method. 相似文献
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Ananya Roy Goutham Ganesh Helena Sippola Sara Bolin Osama Sawesi Anders Dag?lv Susan M. Schlenner Thorsten Feyerabend Hans-Reimer Rodewald Lena Kjellén Lars Hellman Magnus ?brink 《The Journal of biological chemistry》2014,289(1):237-250
During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(Wsash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation. 相似文献
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Michael Murphy Yanbao Xiong Goutham Pattabiraman Fu Qiu Andrei E. Medvedev 《The Journal of biological chemistry》2015,290(31):19218-19232
Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1. 相似文献
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Kato H Nishimura T Ikeda N Yamada T Kondo T Saijo N Nishio K Fujimoto J Nomura M Oda Y Lindmark B Maniwa J Hibino H Unno M Ito T Sawa Y Tojo H Egawa S Edula G Lopez M Wigmore M Inase N Yoshizawa Y Nomura F Marko-Varga G 《Journal of Proteomics》2011,74(6):759-764
Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease that cause mortality, and for that reason are the most active areas for drug development. This perspective paper overviews the urgent need to develop a health care system for a rapidly growing patient population in Japan, including forthcoming demands on clinical care, expecting outcomes, and economics. There is an increasing requirement to build on the strengths of the current health care system, thereby delivering urgent solutions for the future. There is also a declaration from the Ministry of Health, Labour and Welfare (MHLW), to develop new biomarker diagnostics, which is intended for patient stratification, aiding in diagnostic phenotype selection for responders to drug treatment of Japanese patients. This perspective was written by the panel in order to introduce novel technologies and diagnostic capabilities with successful implementation. The next generation of personalized drugs for targeted and stratified patient treatment will soon be available in major disease areas such as, lifestyle-related cancers, especially lung cancers with the highest mortality including a predisposing disorder chronic obstructive pulmonary disease, cardiovascular disease, and other diseases. Mass spectrometric technologies can provide the "phenotypic fingerprint" required for the concept of Personalized Medicine. Mass spectrometry-driven target biomarker diagnoses in combination with high resolution computed tomography can provide a critical pathway initiative facilitated by a fully integrated e-Health infrastructure system. We strongly recommend integrating validated biomarkers based on clinical proteomics, medical imaging with clinical care supported by e-Health model to support personalized treatment paradigms to reduce mortality and healthcare costs of chronic and co-morbid diseases in the elderly population of Japan. 相似文献
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Nagendra S. Punyamurthula Goutham R. Adelli Waseem Gul Michael A. Repka Mahmoud A. ElSohly Soumyajit Majumdar 《AAPS PharmSciTech》2017,18(6):1936-1945
The purposes of this project are to enhance the trans-membrane penetration of Δ8-Tetrahydrocannabinol (Δ8-THC) and to study the effect of various lipid based systems in delivering the compound, non-invasively, to anterior and posterior ocular chambers. Solid lipid nanoparticles (SLNs), fast gelling films were manufactured using high pressure homogenization and melt cast techniques, respectively. The formulations were characterized for drug content, entrapment efficiency, particle size and subsequently evaluated in vitro for trans-corneal permeation. In vivo, the drug disposition was tested via topical administration in albino rabbits. The eye globes were enucleated at the end of experiment and tissues were analyzed for drug content. All formulations showed favorable physicochemical characteristics in terms of particle size, entrapment efficiency, and drug content. In vitro, the formulations exhibited a transcorneal flux that depended on the formulation’s drug load. An increase in drug load from 0.1 to 0.75% resulted in 12- to16-folds increase in permeation. In vivo, the film was able to deliver THC to all the tissues with high accumulations in cornea and sclera. The SLNs showed a greater ability in delivering THC to all the tissues, at a significantly lower drug load, due to their colloidal size range, which in turn enhanced corneal epithelial membrane penetration. The topical formulations evaluated in the present study were able to successfully deliver Δ8-THC in therapeutically meaningful concentrations (EC50 values for CB1: 6 nM and CB2: 0.4 nM) to all ocular tissues except the vitreous humor, with pronounced tissue penetration achieved using SLNs as a Δ8-THC delivery vehicle. 相似文献
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Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator
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Danica D. Wiredja Marzieh Ayati Sahar Mazhar Jaya Sangodkar Sean Maxwell Daniela Schlatzer Goutham Narla Mehmet Koyutürk Mark R. Chance 《Proteomics》2017,17(22)
Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems‐level perturbations mediated by PP2A activation, we carried out mass spectrometry‐based phosphoproteomic analysis of two KRAS mutated non‐small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS‐related signaling are attenuated for A549 compared to H358, while the effects on cell cycle‐related kinases are noticeably more prominent in A549. Network‐based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines. 相似文献
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Patel VA Feng L Lee DJ Massenburg D Pattabiraman G Antoni A Schwartz JH Lieberthal W Rauch J Ucker DS Levine JS 《The Journal of biological chemistry》2012,287(17):13761-13777
Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular β-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses. 相似文献