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Ashley T Fancher, Yun Hua, Daniel P Camarco, David A Close, Christopher J Strock, Paul A Johnston
The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein-protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins...
October 2016: Assay and Drug Development Technologies
Sabari Nath Neerukonda, Upendra K Katneni, Sergey Golovan, Mark S Parcells
Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular β-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a...
October 2016: Journal of Virological Methods
Aggeliki Skagia, Chrysoula Zografou, Eleni Vezyri, Anastasia Venieraki, Panagiotis Katinakis, Maria Dimou
PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC:, which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each ΔppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and ΔppiB genetic backgrounds implying possible post-translational modifications by PpiB...
August 2016: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Dawei Jiang, Yunchao Liu, Aiping Wang, Gaiping Zhang, Guoyu Yang, Yumei Chen, Pengchao Ji, Chang Liu, Yapeng Song, Yunfang Su, Guoqiang Wang, Jucai Wang, Baolei Zhao, Ruiguang Deng
OBJECTIVES: To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli. RESULTS: Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni-NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV...
June 2016: Biotechnology Letters
Saurabh Pandey, Ashish Sharma, Deeksha Tripathi, Ashutosh Kumar, Mohd Khubaib, Manish Bhuwan, Tapan Kumar Chaudhuri, Seyed Ehtesham Hasnain, Nasreen Zafar Ehtesham
Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases...
2016: PloS One
A R Melwani, E L Thompson, D A Raftos
Exposure to prolonged environmental stress can have impacts on the cellular homeostasis of aquatic organisms. The current study employed two-dimensional electrophoresis (2-DE) to test whether exposure to impaired water quality conditions in the Sydney Harbour estuary has significantly altered the proteomes of the resident Sydney rock oyster (Saccostrea glomerata). Adult S. glomerata were sampled from four bays in the estuary. Each bay consisted of a "high-impact" site adjacent to point sources of chemical contamination (e...
April 2016: Aquatic Toxicology
Christian Kirschneck, Peter Proff, Jochen Fanghänel, Michael Wolf, J Camilo Roldán, Piero Römer
OBJECTIVES: To obtain valid results in relative gene/mRNA-expression analyses by RT-qPCR, a careful selection of stable reference genes is required for normalization. Currently there is little information on reference gene stability in dental, periodontal and alveolar bone tissues of the rat, especially regarding orthodontic tooth movement and periodontitis. We therefore aimed to identify the best selection and number of reference genes under these experimental as well as physiological conditions...
March 2016: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
Signe Horn, Jeannette S Kirkegaard, Soraya Hoelper, Philip A Seymour, Claude Rescan, Jens H Nielsen, Ole D Madsen, Jan N Jensen, Marcus Krüger, Mads Grønborg, Jonas Ahnfelt-Rønne
Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies...
January 2016: Molecular Endocrinology
C Barbirato, M Trancozo, M G Almeida, L S Almeida, T O Santos, J C G Duarte, M R G O Rebouças, V Sipolatti, V R R Nunes, F Paula
Osteogenesis imperfecta (OI) is a genetic disease characterized by bone deformities and fractures. Most cases are caused by autosomal dominant mutations in the type I collagen genes COL1A1 and COL1A2; however, an increasing number of recessive mutations in other genes have been reported. The LEPRE1, CRTAP, and PPIB genes encode proteins that form the P3H1/CRTAP/CypB complex, which is responsible for posttranslational modifications of type I collagen. In general, mutations in these genes lead to severe and lethal phenotypes of recessive OI...
2015: Genetics and Molecular Research: GMR
Ibrahim M Shokry, John J Callanan, John Sousa, Rui Tao
3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment...
2015: Journal of Visualized Experiments: JoVE
Pauline Marie, Valérie Labas, Aurélien Brionne, Grégoire Harichaux, Christelle Hennequet-Antier, Alejandro B Rodriguez-Navarro, Yves Nys, Joël Gautron
Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages...
August 3, 2015: Journal of Proteomics
Minghui Liu, Yuchang Dou, Ran Sun, Yonggui Zhang, Yansong Liu
BACKGROUND: Alcoholic hepatitis (AH) is an acute manifestation of alcoholic liver disease with high mortality rates. OBJECTIVES: Our aim was to study the molecular mechanisms of AH. MATERIALS AND METHODS: The differentially expressed genes (DEGs) in liver between AH and control cases were identified by analyzing the GSE28619 microarray data using t-test. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) enrichment analyses were performed using DAVID online tool...
May 2015: Hepatitis Monthly
Yun Hua, Christopher J Strock, Paul A Johnston
This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs...
2015: Methods in Molecular Biology
María Rodríguez-Moyá, Ramon Gonzalez
UNLABELLED: Given their simple and easy-to-manipulate chemical structures, short-chain fatty acids (SCFAs) are valuable feedstocks for many industrial applications. While the microbial production of SCFAs by engineered Escherichia coli has been demonstrated recently, productivity and yields are limited by their antimicrobial properties. In this work, we performed a comparative proteomic analysis of E. coli under octanoic acid stress (15 mM) and identified the underlying mechanisms of SCFA toxicity...
June 3, 2015: Journal of Proteomics
Wuzheng Zhu, Yaqiu Lin, Honghai Liao, Yong Wang
The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development...
2015: PloS One
Renata Moldenhauer Minillo, Nara Sobreira, Maria de Fatima de Faria Soares, Julie Jurgens, Hua Ling, Kurt N Hetrick, Kimberly F Doheny, David Valle, Decio Brunoni, Ana B Alvarez Perez
Autosomal recessive osteogenesis imperfecta (OI) accounts for 10% of all OI cases, and, currently, mutations in 10 genes (CRTAP, LEPRE1, PPIB, SERPINH1, FKBP10, SERPINF1, SP7, BMP1, TMEM38B, and WNT1) are known to be responsible for this form of the disease. PEDF is a secreted glycoprotein of the serpin superfamily that maintains bone homeostasis and regulates osteoid mineralization, and it is encoded by SERPINF1, currently associated with OI type VI (MIM 172860). Here, we report a consanguineous Brazilian family in which multiple individuals from at least 4 generations are affected with a severe form of OI, and we also report an unrelated individual from the same small city in Brazil with a similar but more severe phenotype...
December 2014: Molecular Syndromology
Shahar Molshanski-Mor, Ido Yosef, Ruth Kiro, Rotem Edgar, Miriam Manor, Michael Gershovits, Mia Laserson, Tal Pupko, Udi Qimron
Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterial-growth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes...
December 30, 2014: Proceedings of the National Academy of Sciences of the United States of America
Joshi Stephen, Katta Mohan Girisha, Ashwin Dalal, Anju Shukla, Hitesh Shah, Priyanka Srivastava, Uwe Kornak, Shubha R Phadke
Osteogenesis imperfecta (OI) is a spectrum of genetic disorders with decreased bone density and bone fragility. Most of the cases of OI are inherited in autosomal dominant fashion with mutations in COL1A1 or COL1A2 genes. Over last few years, twelve genes for autosomal recessive OI have been identified. In this study we have evaluated seven patients with OI from consanguineous Indian families. Homozygosity mapping using SNP microarray was done and selected candidate genes were sequenced. Candidate genes were identified in four out of seven patients studied...
January 2015: European Journal of Medical Genetics
YongCheol Yoo, Kyunghee Byun, Taewook Kang, Delger Bayarsaikhan, Jin Young Kim, Seyeoun Oh, Young Hye Kim, Se-Young Kim, Won-Il Chung, Seung U Kim, Bonghee Lee, Young Mok Park
Microglial activation in the central nervous system is a key event in the neuroinflammation that accompanies neurodegenerative diseases such as Alzheimer's disease (AD). Among cytokines involved in microglial activation, amyloid β (Aβ) peptide is known to be a key molecule in the induction of diverse inflammatory products, which may lead to chronic inflammation in AD. However, proteomic studies of microglia in AD are limited due to lack of proper cell or animal model systems. In this study, we performed a proteomic analysis of Aβ-stimulated human microglial cells using SILAC (stable isotope labeling with amino acids in cell culture) combined with LC-MS/MS...
January 2, 2015: Journal of Proteome Research
Yun Hua, Tong Ying Shun, Christopher J Strock, Paul A Johnston
The androgen receptor-transcriptional intermediary factor 2 (AR-TIF2) positional protein-protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) "prey" and TIF2-green fluorescent protein (GFP) "bait" components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence...
September 2014: Assay and Drug Development Technologies
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