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Protein ligation

Lecticia Barbosa Jorge, Fernanda Oliveira Coelho, Talita Rojas Sanches, Denise M A C Malheiros, Leandro Ezaquiel de Souza, Fernando Dos Santos, Larissa de Sá Lima, Cristóforo Scavone, Maria Claudia Irigoyen, Makoto Kuro-O, Lucia Andrade
Sepsis-induced organ failure is characterized by a massive inflammatory response and oxidative stress. Acute kidney injury (AKI) occurs in approximately half of patients in septic shock, and the mortality associated with sepsis-induced AKI is unacceptably high. Klotho is a protein expressed by renal cells and has anti-senescence properties. Klotho has also been shown to protect the kidneys in ischemia-reperfusion injury and to have antioxidant properties. To analyze the role of Klotho in sepsis-related organ dysfunction and AKI, we used a cecal ligation and puncture (CLP) model of sepsis in heterozygous Klotho-haploinsufficient mice and their wild-type littermates (CLP-Kl+/- and CLP-WT mice, respectively)...
December 5, 2018: American Journal of Physiology. Renal Physiology
Ayhan Aköz, Kenan Ahmet Türkdoğan, Nesibe Kahraman Çetin, Selen Kum, Ali Duman, Mevlüt Türe, Ahmet Ender Demirkıran
BACKGROUND: The objective of the current study was to investigate the value of the ischemic biomarkers endothelial cell-specific molecule-1 (endocan) and signal peptide-CUB-EGF domain-containing protein-1 (SCUBE-1) in the diagnosis and assessment of earlystage and irreversible damage in acute mesenteric ischemia. METHODS: An experimental mesenteric ischemia reperfusion model was designed using 54 rats. Nine groups were created: Three sham groups [Groups I (30th minute), IV (2nd hour), and VII (6th hour)], in which only blood and tissue specimens were sampled; 3 ischemia groups [Groups II (30th minute), V (2nd hour), and VIII (6th hour)], in which blood and tissue specimens were sampled after ligation of the superior mesenteric artery (SMA); and 3 reperfusion groups [Groups III (30th minute), VI (2nd hour), and IX (6th hour)], in which blood and tissue specimens were sampled after declamping the SMA and reperfusion for 1 hour...
November 2018: Ulusal Travma Ve Acil Cerrahi Dergisi, Turkish Journal of Trauma & Emergency Surgery: TJTES
Ryota Yamagata, Wataru Nemoto, Osamu Nakagawasai, Wan-Yi Hung, Kazuhiro Shima, Yasuo Endo, Koichi Tan-No
Etidronate is widely used as a therapeutic agent for osteoporosis. We have recently shown that intrathecal administration of etidronate into mice produces an analgesic effect against the capsaicin-induced nociceptive behavior. However, the effect of etidronate on neuropathic pain at the spinal level remains unknown. Therefore, we examined whether etidronate attenuates pain after partial sciatic nerve ligation (PSNL). We evaluated tactile allodynia 7 days after PSNL by measuring paw withdrawal with the von Frey filament test...
December 4, 2018: Naunyn-Schmiedeberg's Archives of Pharmacology
Gonzalo Artiach, Miguel Carracedo, Joan Clària, Andres Laguna-Fernandez, Magnus Bäck
Intimal hyperplasia remains a significant clinical problem in for example coronary artery bypass graft failure. Since omega-3 fatty acids reduce intimal hyperplasia, we hypothesized that the G protein-coupled receptor ChemR23 for the omega-3-derived pro-resolving lipid mediator resolvin E1 drives those effects. ChemR23+/+ and ChemR23-/- mice were generated with or without introduction of the Caenorhabditis elegans fat-1 transgene, which leads to an endogenous omega-3 fatty acid synthesis and thus increasing the substrate for resolvin E1 formation...
2018: Frontiers in Pharmacology
Alice L Baumann, Christian P R Hackenberger
Proteins and peptides are gaining increasing interest as tools and targets in fundamental research and drug discovery. Growing research applications have prompted the need for methodologies that produce homogenous peptide and protein material. The development of efficient, chemoselective ligation reactions using unprotected peptide fragments presents a key solution for this challenging task. This review outlines modern ligation methods that enable the synthesis of both native, and also labelled or post-translationally modified peptides and proteins...
November 30, 2018: Chimia
Adebowale O Bamidele, Phyllis A Svingen, Mary R Sagstetter, Olga F Sarmento, Michelle Gonzalez, Manuel B Braga Neto, Subra Kugathasan, Gwen Lomberk, Raul A Urrutia, William A Faubion
Background & Aims: Forkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function. Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis...
2019: Cellular and Molecular Gastroenterology and Hepatology
Alpna Tyagi, Christy U Nguyen, Thomas Chong, Cole R Michel, Kristofer S Fritz, Nichole Reisdorph, Leslie Knaub, Jane E B Reusch, Subbiah Pugazhenthi
SIRT3, the primary mitochondrial deacetylase, plays a significant role in enhancing the function of mitochondrial proteins. Downregulation of SIRT3 is a key component of metabolic syndrome, a precondition for obesity, diabetes and cardiovascular diseases. In this study, we examined the effects of brain mitochondrial protein hyperacetylation in western diet-fed Sirt3-/- mice, a model for metabolic syndrome. Brain mitochondrial proteins were hyperacetylated, following western diet feeding and Sirt3 deletion. To identity these hyperacetylated proteins, we performed a comprehensive acetylome analysis by label-free tandem mass spectrometry...
December 3, 2018: Scientific Reports
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This protocol describes purification of RNA cross-linking immunoprecipitation (CLIP) tags by proteinase K digestion of the cross-linked protein, addition of a 5' linker to the RNA tags, and amplification of the product by transcription-polymerase chain reaction (RT-PCR). Use of this protocol adds another important purification step: sizing of the PCR products to enrich for those derived from RNA originally cross-linked to the desired RNABP. Finally, sequencing of the PCR products is described. There are two strategies for sequencing the PCR products of "CLIPed" RNA...
December 3, 2018: Cold Spring Harbor Protocols
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3' linker loses the puromycin blocking group, concatemerization of the 3' linker will occur during the 3' linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Additionally, this protocol describes the treatment of coimmunoprecipitated RNA tags for CLIP with alkaline phosphatase to remove the 3' phosphate remaining after RNase digestion...
December 3, 2018: Cold Spring Harbor Protocols
Jennifer C Darnell, Aldo Mele, Ka Ying Sharon Hung, Robert B Darnell
This first part of this protocol is designed to optimize purification of the RNABP by immunoprecipitation for cross-linking immunoprecipitation (CLIP) experiments. The key variables to assess are the quality and quantity of antibody needed to immunoprecipitate most but not quite all of the RNABP (the titration will decrease nonspecific binding), and the tolerance of the antibody:antigen interaction to stringent wash conditions. The results of these experiments can be checked first by western blot, and subsequently using the pilot CLIP protocol described in the second half of this protocol...
December 3, 2018: Cold Spring Harbor Protocols
Emma J Petrie, Peter E Czabotar, James M Murphy
The recent implication of the cell death pathway, necroptosis, in innate immunity and a range of human pathologies has led to intense interest in the underlying molecular mechanism. Unlike the better-understood apoptosis pathway, necroptosis is a caspase-independent pathway that leads to cell lysis and release of immunogens downstream of death receptor and Toll-like receptor (TLR) ligation. Here we review the role of recent structural studies of the core machinery of the pathway, the protein kinases receptor-interacting protein kinase (RIPK)1 and RIPK3, and the terminal effector, the pseudokinase mixed lineage kinase domain-like protein (MLKL), in shaping our mechanistic understanding of necroptotic signaling...
November 30, 2018: Trends in Biochemical Sciences
Guilherme H Gatti da Silva, Melissa S Jurica, Julia P Chagas da Cunha, Carla C Oliveira, Patricia P Coltri
Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity...
December 2, 2018: Journal of Cellular Biochemistry
Lee Ann MacMillan-Crow, Philip R Mayeux
Given the inherent heterogeneity of the septic patient population and possible comorbid conditions, it is not surprising that the influence of gender on incidence and outcomes are still unclear. The goal of this study was to use a clinically relevant murine model of sepsis, cecal ligation and puncture (CLP) in CD1 mice, with and without uniphrectomy as a comorbid condition to investigate possible gender differences in renal mitochondrial function and dynamics. High resolution respirometry on fresh kidney biopsies was used to measure renal respiratory complex activities...
October 2018: Internal Medicine Review (Washington, D.C.: Online)
Takashi Ito, Mayumi Nakahara, Yoshiki Masuda, Sachie Ono, Shingo Yamada, Hiroyasu Ishikura, Hitoshi Imaizumi, Chinatsu Kamikokuryo, Yasuyuki Kakihana, Ikuro Maruyama
Background: Nuclear histone proteins are released into the extracellular space during sepsis and act as major mediators of death. However, circulating histone levels have not been precisely quantified. Methods: We developed a novel enzyme-linked immunosorbent assay (ELISA) for detection of circulating histone H3 levels and evaluated its performance. Using the ELISA, we measured plasma histone H3 levels in C57BL/6 J mice subjected to cecal ligation and puncture (CLP)-induced sepsis...
2018: Journal of Intensive Care
Sha Li, Shuren Li
OBJECTIVE: To evaluate the effects of transplantation of hypoxia-inducible factor-1α (HIF-1α) gene-modified cardiac stem cells (CSCs) on the cardiac function of heart failure rats after myocardial infarction (MI). METHODS: Twenty-four Sprague-Dawley rats were randomly divided into three groups: HIF-1α-modified CSCs group, single CSCs group, and model group. The model of heart failure after MI was established by thoracotomy-left anterior descending coronary artery ligation, followed by injection of modified CSCs, single CSCs, and PBS, respectively, 2 weeks later...
December 2018: Anatolian Journal of Cardiology
Jai J Tree, Kenn Gerdes, David Tollervey
Regulatory RNAs and RNA-binding proteins (RBPs) play critical roles in virulence gene expression in pathogenic bacteria. A wealth of regulatory RNAs have been identified in bacterial pathogens using RNA-seq and recent technical advances are uncovering their mRNA targets. UV-crosslinking is a powerful tool for identifying protein binding sites throughout the transcriptome providing base-pair resolution of sites in vivo. With minor modifications to the protocol, RNA-RNA interactions can also be captured by proximity-dependent ligation of RNA pairs on the protein...
2018: Methods in Enzymology
Zheng-Jie Meng, Chao Wang, Ling-Tong Meng, Bei-Hua Bao, Jin-Hui Wu, Yi-Qiao Hu
Cardiac dysfunction, a common consequence of sepsis, is the major contribution to morbidity and mortality in patients. Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of Tanshinone IIA (TA), a main active component of Salvia miltiorrhiza Bunge, which has been widely used in China for the treatment of cardiovascular and cerebral system diseases. In the present study, the effect of STS on sepsis-induced cardiac dysfunction was investigated and its effect on survival rate of rats with sepsis was also evaluated...
November 2018: Chinese Journal of Natural Medicines
Rodrigo Prado Martins, Sarah Findakly, Chrysoula Daskalogianni, Marie-Paule Teulade-Fichou, Marc Blondel, Robin Fåhraeus
Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers...
November 29, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Fukashi Serizawa, Miyako Tanaka, Takuya Shimizu, Daijirou Akamatsu, Masato Ohara, Hitoshi Goto, Takashi Kamei
OBJECTIVE: Patients presenting with edema, skin redness, pain, and itching in their lower legs are common and encountered often in daily practice. However, although commonly recognized diseases, such as deep venous thrombosis (DVT), stasis dermatitis due to varicose veins, lymphedema, and cellulitis, are diagnosed correctly in most cases, micro-arteriovenous fistulas (AVFs) may often be overlooked due to low awareness and rarity. This study was carried out to evaluate the prevalence of micro-AVF in patients presenting with foot skin symptoms...
November 27, 2018: Annals of Vascular Surgery
Ting Cao, Jing Lv, Lei Zhang, Guoquan Yan, Haojie Lu
The identification and quantification of low abundant proteins are always impeded by high abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. And the quantification was achieved by introducing isotopically labelled threonine in the motif LPXTG...
November 30, 2018: Analytical Chemistry
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