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In a search for anti-inflammatory compounds from fungi inhibiting the promoter activity of the small chemokine CXCL10 (Interferon-inducible protein 10, IP-10) as a pro-inflammatory marker gene, the new dihydroxanthone methyl (1R, 2R)-1,2,8-trihydroxy-6-(hydroxymethyl)-9-oxo-2,9-dihydro-1H-xanthene-1-carboxylate (2) and the previously described dihydroxanthone AGI-B4 (1) were isolated from fermentations of a Diaporthe species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy, mass spectrometry, and calculations using density functional theory (DFT). Compounds 1 and 2 inhibited the LPS/IFNγ induced CXCL10 promoter activity in transiently transfected human MonoMac6 cells in a dose-dependent manner with IC50 values of 4.1 µM (±0.2 µM) and 1.0 µM (±0.06 µM) respectively. Moreover, compounds 1 and 2 reduced mRNA levels and synthesis of pro-inflammatory mediators such as cytokines and chemokines in LPS/IFNγ stimulated MonoMac6 cells by interfering with the Stat1 and NFκB pathway. [ABSTRACT FROM AUTHOR]

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers. [ABSTRACT FROM AUTHOR]

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers. [ABSTRACT FROM AUTHOR]

RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. They contain a variety of chemical modifications and many RNAs fold into complex structures. Here, we review recent progress in the analysis of RNA modification and structure on the basis of stable isotope labeling techniques. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the key tools and many breakthrough developments were made possible by the analysis of stable isotope labeled RNA. Therefore, we discuss current stable isotope labeling techniques such as metabolic labeling, enzymatic labeling and chemical synthesis. RNA structure analysis by NMR is challenging due to two major problems that become even more salient when the size of the RNA increases, namely chemical shift overlaps and line broadening leading to complete signal loss. Several isotope labeling strategies have been developed to provide solutions to these major issues, such as deuteration, segmental isotope labeling or site-specific labeling. Quantification of modified nucleosides in RNA by MS is only possible through the application of stable isotope labeled internal standards. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS), it is now possible to analyze the dynamic processes of post-transcriptional RNA modification and demodification. The trend, in both NMR and MS RNA analytics, is without doubt shifting from the analysis of snapshot moments towards the development and application of tools capable of analyzing the dynamics of RNA structure and modification profiles. [ABSTRACT FROM AUTHOR]

The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation. [ABSTRACT FROM AUTHOR]

Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergentbased reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins. [ABSTRACT FROM AUTHOR]