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intact cell protein profiling

Tongkai Chen, Chuwen Li, Ye Li, Yi Xiang, Simon Ming Yuen Lee, Ying Zheng
Schisantherin A (SA) is a promising anti-Parkinsonism Chinese herbal medicine but with poor water solubility and challenges to be delivered to the brain. We formulated SA as nanocrystals (SA-NC), aiming to improve its solubility, pharmacokinetic profile and thus provide a potential therapeutic agent for the treatment of Parkinson's disease (PD). The rod-shaped SA-NC had a particle size of ~160 nm with 33.3% drug loading, and the nanocrystals exhibited a fast dissolution rate in vitro. The intact drug nanocrystals could be internalized into Madin-Darby canine kidney (MDCK) cells, which were followed by rapid intracellular release, and most of the drug was transported to the basolateral side in its soluble form...
October 14, 2016: Molecular Pharmaceutics
Simon B M Kretschmer, Stefano Woltersdorf, Dominik Vogt, Felix F Lillich, Michael Rühl, Michael Karas, Isabelle V Maucher, Jessica Roos, Ann-Kathrin Häfner, Astrid Kaiser, Mario Wurglics, Manfred Schubert-Zsilavecz, Carlo Angioni, Gerd Geisslinger, Holger Stark, Dieter Steinhilber, Bettina Hofmann
5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we present the detailed molecular mechanism of action of the compound class and the in vitro pharmacological profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant proteins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with unique characteristics...
September 23, 2016: Biochemical Pharmacology
Michael H Heggeness, Nora Strong, Paul H Wooley, Shang-You Yang
BACKGROUND: The clinical use of recombinant human bone morphogenic protein 2 (rhBMP-2, Infuse) has been associated with nerve-related complications including new-onset sciatica, and retrograde ejaculation. PURPOSE: To better understand the interaction of rhBMP-2 and peripheral nerves with the intent of making procedures safer. STUDY DESIGN/SETTING: Using a mouse model to examine the direct effect of diluted rhBMP-2 (Infuse) on murine sciatic nerves...
September 21, 2016: Spine Journal: Official Journal of the North American Spine Society
Lina Zhang, Giacomo Lanzoni, Matteo Battarra, Luca Inverardi, Qibin Zhang
: The etiology of Type 1 Diabetes (T1D) remains elusive. Enzymatically isolated and cultured (EIC) islets cannot fully reflect the natural protein composition and disease process of in vivo islets, because of the stress from isolation procedures. In order to study islet protein composition in conditions close to the natural environment, we performed proteomic analysis of EIC islets, and laser capture microdissected (LCM) human islets and acinar tissue from fresh-frozen pancreas sections of three cadaveric donors...
September 13, 2016: Journal of Proteomics
Norbert W Lutz, Pallavi Banerjee, Brian J Wilson, Jie Ma, Patrick J Cozzone, Markus H Frank
We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids...
2016: PloS One
Ling-Lei Kong, Guo-Lin Shen, Zhi-Yuan Wang, Xiao-Mei Zhuang, Wei-Bin Xiao, Mei Yuan, Ze-Hui Gong, Hua Li
Thienorphine (TNP) is a novel partial opioid agonist that has completed phase II clinical evaluation as a promising drug candidate for the treatment of opioid dependence. Previous studies have shown that TNP and its glucuronide conjugate (TNP-G) undergo significant bile excretion. The purpose of this study was to investigate the roles of efflux transporters in regulating biliary excretion and plasma exposure of TNP and TNP-G. An ATPase assay suggested that TNP and TNP-G were substrates of P-gp and MRP2, respectively...
2016: Frontiers in Pharmacology
Barry W Festoff, Ravi K Sajja, Patrick van Dreden, Luca Cucullo
BACKGROUND: The blood-brain barrier (BBB) dysfunction represents an early feature of Alzheimer's disease (AD) that precedes the hallmarks of amyloid beta (amyloid β) plaque deposition and neuronal neurofibrillary tangle (NFT) formation. A damaged BBB correlates directly with neuroinflammation involving microglial activation and reactive astrogliosis, which is associated with increased expression and/or release of high-mobility group box protein 1 (HMGB1) and thrombin. However, the link between the presence of these molecules, BBB damage, and progression to neurodegeneration in AD is still elusive...
2016: Journal of Neuroinflammation
Jana Beinhauer, René Lenobel, Dmitry Loginov, Ivo Chamrád, Pavel Řehulka, Michaela Sedlářová, Martina Marchetti-Deschmann, Günter Allmaier, Marek Šebela
Several proteomic approaches were applied to identify protein markers providing typical signals during intact cell/spore (IC/IS) MALDI-TOF MS of two plant pathogens, namely Bremia lactucae (a downy mildew) and Oidium neolycopersici (a powdery mildew). First, proteins were extracted from intact spores of the microorganisms under conditions simulating their treatment prior to the mass spectrometric analysis. After a separation by electrophoresis and tryptic digestion, 198 and 140 proteins were identified in the B...
August 23, 2016: Electrophoresis
Hua Yang, Weidong Chen, Jiahao Shi, Dandan Huang, Jun Li, Binyang Hu, Mengjie Zhang, Zhugang Wang, Jian Fei
Abi3bp was originally discovered as Abi3-Src homology 3 (SH3) binding protein and has been proved to have a broad expression profile in adult tissues. Although previous studies have indicated that Abi3bp may be associated with cancer suppression, cell senescence, dendritic refinement and mental disorder, most conclusions achieved were based on in vitro model or genome-wide association study. In this work, we constructed an Abi3bp-deficient mouse model and observed phenotypic changes. The generated Abi3bp-knockout mice are viable and fertile, develop normally and exhibit no significant differences in anxiety or depression-like behaviors, olfactory function and tumor incidence...
September 2016: Acta Biochimica et Biophysica Sinica
Pei-Jing Pai, Yingwei Hu, Henry Lam
Intact glycopeptide MS analysis to reveal site-specific protein glycosylation is an important frontier of proteomics. However, computational tools for analyzing MS/MS spectra of intact glycopeptides are still limited and not well-integrated into existing workflows. In this work, a new computational tool which combines the spectral library building/searching tool, SpectraST (Lam et al. Nat. Methods2008, 5, 873-875), and the glycopeptide fragmentation prediction tool, MassAnalyzer (Zhang et al. Anal. Chem.2010, 82, 10194-10202) for intact glycopeptide analysis has been developed...
August 31, 2016: Analytica Chimica Acta
Joel D Federspiel, Simona G Codreanu, Sandeep Goyal, Matthew E Albertolle, Eric Lowe, Juli Teague, Hansen Wong, F Peter Guengerich, Daniel C Liebler
Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the β5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction...
October 2016: Molecular & Cellular Proteomics: MCP
Christian Niehage, Jana Karbanová, Charlotte Steenblock, Denis Corbeil, Bernard Hoflack
Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down...
2016: PloS One
Takehiro Shinoda, Naoko Shinya, Kaori Ito, Yoshiko Ishizuka-Katsura, Noboru Ohsawa, Takaho Terada, Kunio Hirata, Yoshiaki Kawano, Masaki Yamamoto, Taisuke Tomita, Yohei Ishibashi, Yoshio Hirabayashi, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama
The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits...
2016: Scientific Reports
Sriram Vaidyanathan, Bradford G Orr, Mark M Banaszak Holl
Cationic polymers have been investigated as nonviral vectors for gene delivery due to their favorable safety profile when compared to viral vectors. However, nonviral vectors are limited by poor efficacy in inducing gene expression. The physicochemical properties of cationic polymers enabling successful gene expression have been investigated in order to improve expression efficiency and safety. Studies over the past several years have focused on five possible rate-limiting processes to explain the differences in gene expression: (1) endosomal release, (2) transport within specific intracellular pathways, (3) protection of DNA from nucleases, (4) transport into the nucleus, and (5) DNA release from vectors...
August 16, 2016: Accounts of Chemical Research
Jasmine C C Davis, Sarah M Totten, Julie O Huang, Sadaf Nagshbandi, Nina Kirmiz, Daniel A Garrido, Zachery T Lewis, Lauren D Wu, Jennifer T Smilowitz, J Bruce German, David A Mills, Carlito B Lebrilla
Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population...
September 2016: Molecular & Cellular Proteomics: MCP
Vlasta Korenkova, Jana Slyskova, Vendula Novosadova, Sara Pizzamiglio, Lucie Langerova, Jens Bjorkman, Ondrej Vycital, Vaclav Liska, Miroslav Levy, Karel Veskrna, Pavel Vodicka, Ludmila Vodickova, Mikael Kubista, Paolo Verderio
Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling...
2016: Scientific Reports
P L Martin, J-J Yin, V Seng, O Casey, E Corey, C Morrissey, R M Simpson, K Kelly
Prostate cancer is characterized by a dependence upon androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) is the accepted treatment for progressive prostate cancer. Although ADT is usually initially effective, acquired resistance termed castrate-resistant prostate cancer (CRPC) develops. PTEN and TP53 are two of the most commonly deleted or mutated genes in prostate cancer, the compound loss of which is enriched in CRPC. To interrogate the metabolic alterations associated with survival following ADT, we used an orthotopic model of Pten/Tp53 null prostate cancer...
July 4, 2016: Oncogene
Joise Hander Mares, Karina Peres Gramacho, Everton Cruz Dos Santos, André da Silva Santiago, Edson Mário de Andrade Silva, Fátima Cerqueira Alvim, Carlos Priminho Pirovani
BACKGROUND: Witches' broom, a disease caused by the basidiomycete Moniliophthora perniciosa, is considered to be the most important disease of the cocoa crop in Bahia, an area in the Brazilian Amazon, and also in the other countries where it is found. M. perniciosa germ tubes may penetrate into the host through intact or natural openings in the cuticle surface, in epidermis cell junctions, at the base of trichomes, or through the stomata. Despite its relevance to the fungal life cycle, basidiospore biology has not been extensively investigated...
2016: BMC Microbiology
Petra Talapka, Anikó Berkó, Lajos István Nagy, Lalitha Chandrakumar, Mária Bagyánszki, László Géza Puskás, Éva Fekete, Nikolett Bódi
AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations...
June 14, 2016: World Journal of Gastroenterology: WJG
Reut Falach, Anita Sapoznikov, Yoav Gal, Ofir Israeli, Moshe Leitner, Nehama Seliger, Sharon Ehrlich, Chanoch Kronman, Tamar Sabo
The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template...
September 6, 2016: Toxicology Letters
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