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Katoh A A Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; Otorhynolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; Department of Otorhynolaryngology, National Kyushu Medical Center, Fukuoka, - - 2014
The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time ...
Besnard Fabrice F Laboratoire de Reproduction et Développement des Plantes; CNRS; INRA; ENS Lyon; UCBL; Université de Lyon; Lyon, - - 2014
Phyllotaxis, the spatio-temporal pattern of organogenesis at the shoot apical meristem, emerges in large part from inhibitory fields consisting in auxin-depleted areas centered on organs. We recently demonstrated the existence of an additional hormone-based inhibitory field generated by Arabidopsis Histidine Phosphotransfer Protein 6 (AHP6), an inhibitor of cytokinin signaling. We ...
Almeida Alexandra D AD Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY , - - 2014
The ability to image cells live and in situ as they proliferate and differentiate has proved to be an invaluable asset to biologists investigating developmental processes. Here, we describe a Spectrum of Fates approach that allows the identification of all the major neuronal subtypes in the zebrafish retina simultaneously. Spectrum ...
Kovaliov Marina M Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, - - 2014
A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating ...
Törner Kerstin K Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, - - 2014
The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method ...
Grimm Verena V Institute of Microbiology and Biotechnology, University of Ulm, 89068 Ulm, - - 2014
Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria has hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonisation of, and interaction with ...
Borg Sarah S Ludwig Maximillians University Munich, Department of Biology I, Martinsried, - - 2014
The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physico-chemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression ...
Amin Marli M Department of Obstetrics and Gynecology (M.A., A.S., M.C., P.S., G.D.C., D.A.D.), David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90024; Department of Medicine Statistics Core (T.G., D.E.), David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, Los Angeles, California 90095; Quest Diagnostics Nichols Institute (N.J.C.), San Juan Capistrano, California 92675; and ART Reproductive Center (C.B.-J., D.H.), Beverly Hills, California - - 2014
Context: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown. Objective: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA ...
Anderson Eric G EG Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, - - 2014
Single molecule assays of splicing and spliceosome assembly can provide unique insights into pre-mRNA processing that complement other technologies. Key to these experiments is the fabrication of fluorescent molecules (pre-mRNAs and spliceosome components) and passivated glass slides for each experiment. Here we describe how to produce fluorescent RNAs by splinted ...
Ko Hae Young HY Department of Biomedical Science, College of Life Science, CHA University, 605-21 Yeoksam 1-dong, Gangnam-gu, Seoul 135-081, Republic of - - 2014
A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA ...
Sinnamon John R JR Center for Nervous Systems Disorders, Centers for Molecular Medicine, Stony Brook University, Stony Brook, New York 11749, - - 2013
The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to ...
Nie Yu-Qiang - - 2013
Accumulating evidence supports the role of miR-122 in fatty liver disease. We investigated miR-122 expression in a steatotic hepatocyte model, the effect of miR-122 over-expression and inhibition in the pathogenesis. Human hepatic cell line L02 was induced with oleic acid to establish the steatotic hepatocyte model. Intracellular lipid content was ...
Gasnier Maxime - - 2013
RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators ...
Patel Asha Parbhu - - 2013
SUMMARY Green fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in several Leishmania species, mostly for in vitro ...
Nawroth Isabel - - 2013
The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both ...
Siedler Solvej - - 2013
This study describes the construction of two flavonoid biosensors, which can be applied for metabolic engineering of Escherichia coli strains. The biosensors are based on transcriptional regulators combined with autofluorescent proteins. The transcriptional activator FdeR from Herbaspirillum seropedicae SmR1 responds to naringenin, while the repressor QdoR from Bacillus subtilis is ...
Bent Zachary W - - 2013
Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration ...
McFarland Karen N - - 2013
Transcriptional dysregulation has been proposed to play a major role in the pathology of Huntington's disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we ...
Mueller Florian - - 2013
Abstract Transcription factors (TFs) interact dynamically in vivo with chromatin binding sites. Here we summarize and compare the four different techniques that are currently used to measure these kinetics in live cells, namely fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single molecule tracking (SMT) and competition ChIP (CC). ...
Pothoulakis Georgios - - 2013
Characterisation of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterisation of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the ...
Höfer Katharina - - 2013
In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time ...
Appleby Sarah L - - 2013
It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the ...
Kim So Yeon - - 2013
Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy ...
Onishi Hiromasa - - 2013
Expression of scFv in Brevibacillus choshinensis was tested using combinations of three different promoters and four different secretion signals. Two model scFv constructs, i.e., His-scFvFLU and His-scFvHEL, were successfully expressed with some of the combinations. Ni Sepharose column and size exclusion chromatography resulted in fairly pure preparations of these two ...
Ferent Julien - - 2013
The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental ...
Liang Shuli - - 2013
The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P GCW14 , and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P GCW14 were cloned upstream of the EGFP ...
Ryoo Soo-Ryoon - - 2013
MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. ...
Rosales Tilman - - 2013
Myc levels are highly regulated and usually low in vivo. Dimerized with Max, it regulates most expressed genes and so directly and indirectly controls most cellular processes. Intranuclear diffusion of a functional c-Myc-eGFP, expressed from its native locus in murine fibroblasts and 3T3 cells or by transient transfection, was monitored ...
Segal Meirav - - 2013
Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red shifted as compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and cyclin ...
Skinner Samuel O SO Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, - - 2013
We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent ...
Zheng Sika - - 2013
Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening ...
Na Yu-Ran - - 2013
Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 ...
Kashyap Neha - - 2013
MicroRNAs (miRNAs) are a class of short noncoding RNAs which are endogenously expressed in many organisms and regulate gene expression by binding to messenger RNA (mRNA). MicroRNAs are either produced from their independent transcription units in intergenic regions or lie in intragenic regions. Intragenic miRNAs and their host mRNAs are ...
Zhang Houshuang - - 2013
Detection of Toxoplasma gondii conversion from the tachyzoite stage to the bradyzoite stage in living brain tissue is difficult because the parasites are small and conversion and reactivation of the parasites are transient events. To better understand the mechanisms of T. gondii stage conversion between tachyzoites and bradyzoites, and to ...
Fang Wei - - 2013
Transgenic animals are powerful tools to study gene function in vivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCR(RH2)-RH2-1 or LCR(RH2)-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCR(RH2)-RH2-1:GFP)(pt112) and ...
Vijftigschild Lodewijk A W - - 2013
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on ...
Das Sadhan - - 2013
Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate ...
Wu Ping - - 2013
RNA interference (RNAi) has emerged as a powerful tool in basic research and therapeutics by silencing the expression of specific target genes. RNAi is triggered by double-stranded small interfering RNAs, which can be processed from naturally or artificially expressed microRNAs (miRNAs) or small hairpin RNAs (shRNAs). The development of reliable ...
Mishra Saurabh - - 2013
The present study was designed to determine the half-life of gfp(m) (2+) mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP(m) (2+) protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp(m) (2+), than the fluorescence intensity of ...
Foo Lynette C - - 2013
The purification of astrocytes by fluorescence-activated cell sorting (FACS) requires that an astrocyte-specific promoter drive the expression of the green fluorescent protein (GFP). Our laboratory uses FACS to acutely isolate astrocytes from young and old tissue as well as to isolate GFP-negative neurons at the end of the FACS sorting ...
McIsaac R Scott - - 2013
The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made ...
Ferguson Matthew L - - 2013
Single-cell studies of gene regulation suggest that transcription dynamics play a fundamental role in determining expression heterogeneity within a population. In addition, the three-dimensional organization of the nucleus seems to both reflect and influence expression patterns in the cell. Therefore, to gain a holistic understanding of transcriptional regulation, it is ...
Hliscs Marion - - 2013
Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of ...
Harada Kenu - - 2013
Transcription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based ...
Koo B - - 2012
The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign ...
Amore Antonella - - 2012
In silico analyses of several laccase promoter sequences have shown the presence of many different responsive elements differentially distributed along the promoter sequences. Analysis of P. ostreatus laccase promoter poxa1b extending around 1400-bp upstream of the start codon showed the presence of several putative response elements, such as ten Metal ...
Nguyen Khac-Hung - - 2012
Both inferior turbinate hypertrophy and nasal polyp formation entail the enlargement of the nasal mucosa caused by rhinosinusitis, but their macro/microscopic and clinical findings differ markedly. This study aimed at investigating differences in the expressions of erbB1/2 and the tight junction proteins, claudin-1 and tricellulin, in the two tissues. Ten ...
Cockwell Heather - - 2012
OBJECTIVE: The current focus of kisspeptin research is an exploration of its key role in the hypothalamic control of human and animal fertility. Notwithstanding the importance of these studies, strong evidence exists that the gene encoding human kisspeptin, KISS1, is present in several peripheral sites, including the placenta. We also ...
Castilla-Llorente Virginia - - 2012
The microRNA (miRNA)-induced silencing complex (miRISC) controls gene expression by a posttranscriptional mechanism involving translational repression and/or promoting messenger RNA (mRNA) deadenylation and degradation. The GW182/TNRC6 (GW) family proteins are core components of the miRISC and are essential for miRNA function. We show that mammalian GW proteins have distinctive functions ...
Mortensen Sussi B - - 2012
Adipocyte renewal from preadipocytes has been shown to occur throughout life and to contribute to obesity, yet very little is known about the molecular circuits that control preadipocyte expansion. The soluble form of the preadipocyte factor (also known as pref-1) delta-like 1 homolog (DLK1(S)) is known to inhibit adipogenic differentiation; ...
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