Identification of hyperoside metabolites in rat using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry

In this paper, ultra performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF) with automated data analysis software (Metabolynx?) were applied for fast analysis of hyperoside metabolites in rat after intravenous administration. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 metabolites in rat plasma, urine and bile. The results indicated that methylation, sulfation and glucuronidation were the major metabolic pathways of hyperoside in vivo, and among them, 3'-O-methyl-hyperoside was confirmed by matching its fragmentation patterns with standard compound. The present study provided important information about the metabolism of hyperoside which will be helpful for fully understanding the mechanism of this compound's action. Furthermore, this work demonstrated the potential of the UPLC/QTOFMS approach using Metabolynx for fast and automated identification of metabolites of natural product.     

A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

A survey of the geographic distribution of Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the best known fungi in Traditional Chinese Medicine. Many efforts have been devoted to locating the production areas of this species resulting in various reports; however, its geographic distribution remains incompletely understood. Distribution of O. sinensis at the county level is clarified in this work based on both a literature search and fieldwork. More than 3600 publications related to O. sinensis were investigated, including scientific papers, books, and online information. Herbarium specimens of O. sinensis and field collections made by this research group during the years 2000-2010 were examined to verify the distribution sites. A total of 203 localities for O. sinensis have been found, of which 106 are considered as confirmed distribution sites, 65 as possible distribution sites, 29 as excluded distribution sites and three as suspicious distribution sites. The results show that O. sinensis is confined to the Tibetan Plateau and its surrounding regions, including Tibet, Gansu, Qinghai, Sichuan, and Yunnan provinces in China and in certain areas of the southern flank of the Himalayas, in the countries of Bhutan, India and Nepal, with 3,000 m as the lowest altitude for the distribution. The fungus is distributed from the southernmost site in Yulong Naxi Autonomous County in northwestern Yunnan Province to the northernmost site in the Qilian Mountains in Qilian County, Qinghai Province, and from the east edge of the Tibetan Plateau in Wudu County, Gansu Province to the westernmost site in Uttarakhand, India. The clarification of the geographic distribution of O. sinensis will lay the foundation for conservation and sustainable use of the species.     

Plant lectins: targeting programmed cell death pathways as antitumor agents

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.     

Detection of advanced oxidation protein products in patients with chronic kidney disease by a novel monoclonal antibody

Advanced oxidation protein products (AOPP) as a biomarker of oxidative stress has been demonstrated in chronic kidney disease (CKD) patients; however, current methods to detect the accumulation of AOPP in serum and in tissues are limited and unreliable. This study generated a monoclonal antibody (mAb) designated 3F2, that reacts specifically with hypochlorous acid (HOCl)-modified proteins, but not with the native forms or with other types of oxidative modifications. Notably, mAb 3F2 recognizes the AOPP deposited in renal tissues of AOPP-treated rats and of patients with different kinds of CKD. Moreover, this mAb can almost completely inhibit the production of reactive oxygen species in RAW264.7 cells induced by AOPP (p < 0.001). In conclusion, mAb 3F2 can be used to detect AOPP specifically in serum and in tissues, and this antibody can potentially provide an important tool and new insight into research on diseases related to oxidative stress.     

Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (P<0.01) in HPL group. HPL contains PDGF (platelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.     

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.