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intact cell protein profiling

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https://www.readbyqxmd.com/read/28213525/conformational-profiling-of-the-at1-angiotensin-ii-receptor-reflects-biased-agonism-g-protein-coupling-and-cellular-context
#1
Dominic Devost, Rory Sleno, Darlaine Petrin, Alice Zhang, Yuji Shinjo, Rakan Okde, Junken Aoki, Asuka Inoue, Terence E Hebert
Here, we report the design and use of GPCR-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use Bioluminescence Resonance Energy Transfer (BRET) with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail and the small fluorescent molecule FlAsH, as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and carboxy-terminal tail of the angiotensin II type I receptor (AT1R)...
February 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28158294/deep-proteome-mapping-of-wm-266-4-human-metastatic-melanoma-cells-from-oncogenic-addiction-to-druggable-targets
#2
Eumorphia G Konstantakou, Athanassios D Velentzas, Athanasios K Anagnostopoulos, Zoi I Litou, Ourania A Konstandi, Aikaterini F Giannopoulou, Ema Anastasiadou, Gerassimos E Voutsinas, George Th Tsangaris, Dimitrios J Stravopodis
Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis. Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy. Metastatic melanoma's heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease...
2017: PloS One
https://www.readbyqxmd.com/read/28151945/antigenic-characterization-of-the-human-immunodeficiency-virus-hiv-1-envelope-glycoprotein-precursor-incorporated-into-nanodiscs
#3
Kristen C Witt, Luis Castillo-Menendez, Haitao Ding, Nicole Espy, Shijian Zhang, John C Kappes, Joseph Sodroski
The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development...
2017: PloS One
https://www.readbyqxmd.com/read/28145808/characterization-of-the-l-glutamate-clearance-pathways-across-the-blood-brain-barrier-and-the-effect-of-astrocytes-in-an-in%C3%A2-vitro-blood-brain-barrier-model
#4
Hans Cc Helms, Blanca I Aldana, Simon Groth, Morten M Jensen, Helle S Waagepetersen, Carsten U Nielsen, Birger Brodin
The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co-application of transporter inhibitors and competing amino acids. Endothelial abluminal L-glutamate uptake was almost abolished by co-application of an EAAT-1 specific inhibitor, whereas luminal uptake was inhibited by L-glutamate and L-aspartate (1 mM)...
January 1, 2017: Journal of Cerebral Blood Flow and Metabolism
https://www.readbyqxmd.com/read/28130393/irradiation-affects-germ-and-somatic-cells-in-prepubertal-monkey-testis-xenografts
#5
I Tröndle, B Westernströer, J Wistuba, N Terwort, S Schlatt, N Neuhaus
STUDY QUESTION: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? SUMMARY ANSWER: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. WHAT IS KNOWN ALREADY: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation...
January 27, 2017: Molecular Human Reproduction
https://www.readbyqxmd.com/read/28109117/lipid-profiling-of-parkin-mutant-human-skin-fibroblasts
#6
Simona Lobasso, Paola Tanzarella, Daniele Vergara, Michele Maffia, Tiziana Cocco, Angela Corcelli
Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein...
January 21, 2017: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/28095896/screening-for-potential-genes-associated-with-bone-overgrowth-after-mid-shaft-femur-fracture-in-a-rat-model
#7
Chibing Liu, Yanting Liu, Weizhong Zhang, Xiuxin Liu
BACKGROUND: We investigated the underlying molecular mechanisms of bone overgrowth after femoral fracture by using high-throughput bioinformatics approaches. METHODS: The gene expression profile of GSE3298 (accession number) was obtained from the Gene Expression Omnibus database. Sixteen femoral growth plate samples, including nine samples without fracture and seven fracture samples for seven time points, were used for analysis. The Limma package was applied to identify differentially expressed genes (DEGs) between fractured and intact samples...
January 17, 2017: Journal of Orthopaedic Surgery and Research
https://www.readbyqxmd.com/read/28011869/pcom-db-update-a-protein-co-migration-database-for-photosynthetic-organisms
#8
Atsushi Takabayashi, Saeka Takabayashi, Kaori Takahashi, Mai Watanabe, Hiroko Uchida, Akio Murakami, Tomomichi Fujita, Masahiko Ikeuchi, Ayumi Tanaka
The identification of protein complexes is important to understand protein structure and function, and the regulation of cellular processes. We used blue-native PAGE and tandem mass spectrometry to identify protein complexes systematically, and built a web database, the protein co-migration database (PCoM-DB, http://pcomdb.lowtem.hokudai.ac.jp/proteins/top), to provide prediction tools for protein complexes. PCoM-DB provides migration profiles for any given protein of interest, and allows users to compare them with migration profiles of other proteins, showing the oligomeric states of proteins and thus identifying potential interaction partners...
December 22, 2016: Plant & Cell Physiology
https://www.readbyqxmd.com/read/28001444/vitamin-d-receptor-expression-in-plasmablastic-lymphoma-and-myeloma-cells-confers-susceptibility-to-vitamin-d
#9
Duncan M Gascoyne, Linden Lyne, Hayley Spearman, Francesca M Buffa, Elizabeth J Soilleux, Alison H Banham
Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphoma (DLBCL). These diseases can be difficult to diagnose and treat, and lack well-characterised cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORα, IGF1 and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumour cells, and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyses active vitamin D3 production...
December 21, 2016: Endocrinology
https://www.readbyqxmd.com/read/27986537/meaningful-respirometric-measurements-of-ucp1-mediated-thermogenesis
#10
REVIEW
Yongguo Li, Tobias Fromme, Martin Klingenspor
Regarding the enormous interest in brown and brite/beige adipose tissue in the context of metabolic disease, reliable quantification of thermogenesis in these adipocytes is a central issue. This requires an assay specific for uncoupling protein 1 (UCP1) mediated thermogenesis in adherent intact cells. In a recent study we identified a major pitfall associated with established procedures generally applied for this purpose. Meaningful respirometry of UCP1-mediated thermogenesis imperatively requires activation of UCP1 and control over free fatty acid levels...
March 2017: Biochimie
https://www.readbyqxmd.com/read/27910159/staphylococcus-aureus-derived-membrane-vesicles-exacerbate-skin-inflammation-in-atopic-dermatitis
#11
S H Jun, J H Lee, S I Kim, C W Choi, T I Park, H R Jung, J W Cho, S H Kim, J C Lee
BACKGROUND: Skin colonization or infection with Staphylococcus aureus is known to trigger aggravation of atopic dermatitis (AD). However, the exact mechanisms by which S. aureus can worsen AD are unknown. OBJECTIVE: We investigated whether and how S. aureus-derived membrane vesicles (MVs) contribute to worsening of AD. METHODS: Immunohistochemical and immunoelectron microscopic analyses were performed to detect staphylococcal protein A (SPA) in the epidermis of AD lesions...
January 2017: Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology
https://www.readbyqxmd.com/read/27813271/beyond-quantification-in-situ-analysis-of-transcriptome-and-pre-mrna-alternative-splicing-at-the-nanoscale
#12
REVIEW
Yi Cui, Jing Liu, Joseph Irudayaraj
In situ analysis offers a venue for dissecting the complex transcriptome in its natural context to tap into cellular processes that could explain the phenotypic physiology and pathology yet to be understood. Over the past decades, enormous progress has been made to improve the resolution, sensitivity, and specificity of single-cell technologies. The continued efforts in RNA research not only facilitates mechanistic studies of molecular biology but also provides state-of-the-art strategies for diagnostic purposes...
November 4, 2016: Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology
https://www.readbyqxmd.com/read/27740776/oral-delivery-of-a-nanocrystal-formulation-of-schisantherin-a-with-improved-bioavailability-and-brain-delivery-for-the-treatment-of-parkinson-s-disease
#13
Tongkai Chen, Chuwen Li, Ye Li, Xiang Yi, 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 and 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...
November 7, 2016: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/27671344/characterization-of-the-molecular-mechanism-of-5-lipoxygenase-inhibition-by-2-aminothiazoles
#14
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...
January 1, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/27664339/quiescent-pluripotent-stem-cells-reside-within-murine-peripheral-nerves-that-can-be-stimulated-to-proliferate-by-recombinant-human-bone-morphogenic-protein-2-or-by-nerve-trauma
#15
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
https://www.readbyqxmd.com/read/27620696/proteomic-profiling-of-human-islets-collected-from-frozen-pancreata-using-laser-capture-microdissection
#16
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...
January 6, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/27560924/expression-of-cell-surface-marker-abcb5-causes-characteristic-modifications-of-glucose-amino-acid-and-phospholipid-metabolism-in-the-g3361-melanoma-initiating-cell-line
#17
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
https://www.readbyqxmd.com/read/27555820/inhibition-of-p-glycoprotein-and-multidrug-resistance-associated-protein-2-regulates-the-hepatobiliary-excretion-and-plasma-exposure-of-thienorphine-and-its-glucuronide-conjugate
#18
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
https://www.readbyqxmd.com/read/27553758/hmgb1-and-thrombin-mediate-the-blood-brain-barrier-dysfunction-acting-as-biomarkers-of-neuroinflammation-and-progression-to-neurodegeneration-in-alzheimer-s-disease
#19
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
https://www.readbyqxmd.com/read/27550208/identification-of-bremia-lactucae-and-oidium-neolycopersici-proteins-extracted-for-intact-spore-maldi-mass-spectrometric-biotyping
#20
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...
November 2016: Electrophoresis
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