Live visualization of genomic loci with BiFC-TALE.

PubMed

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

PubMed Central

Cooper, Simon E; Hodimont, Elsie; Green, Catherine M

2015-01-01

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes. PMID:26030842

Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.

PubMed

Kaufmann, Christine; Sauter, Margret

2017-01-01

Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

PubMed

Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret

2012-08-01

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Effect of Resveratrol, a SIRT1 Activator, on the Interactions of the CLOCK/BMAL1 Complex

PubMed Central

Park, Insung; Lee, Yool; Kim, Hee-Dae

2014-01-01

Background In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression. Methods To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression. Results BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes. Conclusion These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity. PMID:25309798

Interaction study of rice stripe virus proteins reveals a region of the nucleocapsid protein (NP) required for NP self-interaction and nuclear localization.

PubMed

Lian, Sen; Cho, Won Kyong; Jo, Yeonhwa; Kim, Sang-Min; Kim, Kook-Hyung

2014-04-01

Rice stripe virus (RSV), which belongs to the genus Tenuivirus, is an emergent virus problem. The RSV genome is composed of four single-strand RNAs (RNA1-RNA4) and encodes seven proteins. We investigated interactions between six of the RSV proteins by yeast-two hybrid (Y2H) assay in vitro and by bimolecular fluorescence complementation (BiFC) in planta. Y2H identified self-interaction of the nucleocapsid protein (NP) and NS3, while BiFC revealed self-interaction of NP, NS3, and NCP. To identify regions(s) and/or crucial amino acid (aa) residues required for NP self-interaction, we generated various truncated and aa substitution mutants. Y2H assay showed that the N-terminal region of NP (aa 1-56) is necessary for NP self-interaction. Further analysis with substitution mutants demonstrated that additional aa residues located at 42-47 affected their interaction with full-length NP. These results indicate that the N-terminal region (aa 1-36 and 42-47) is required for NP self-interaction. BiFC and co-localization studies showed that the region required for NP self-interaction is also required for NP localization at the nucleus. Overall, our results indicate that the N-terminal region (aa 1-47) of the NP is important for NP self-interaction and that six aa residues (42-47) are essential for both NP self-interaction and nuclear localization. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural features of the KPI domain control APP dimerization, trafficking, and processing.

PubMed

Ben Khalifa, Naouel; Tyteca, Donatienne; Marinangeli, Claudia; Depuydt, Mathieu; Collet, Jean-François; Courtoy, Pierre J; Renauld, Jean-Christophe; Constantinescu, Stefan; Octave, Jean-Noël; Kienlen-Campard, Pascal

2012-02-01

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

PubMed

Wang, Sheng; Chen, Xuanze; Chang, Lei; Ding, Miao; Xue, Ruiying; Duan, Haifeng; Sun, Yujie

2018-06-05

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques.

PubMed

Kitamura, Akira; Nagata, Kazuhiro; Kinjo, Masataka

2015-03-16

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

A Novel System for Visualizing Alphavirus Assembly

PubMed Central

Steel, J. Jordan; Geiss, Brian J.

2015-01-01

Alphaviruses are small, enveloped RNA viruses that form infectious particles by budding through the cellular plasma membrane. To help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (BiFC) that allows visualization of capsid and E2 subcellular localization and association in live cells. In this system, N- or C-terminal Venus fluorescent protein fragments (VN- and VC-) are fused to the N-terminus of the capsid protein on the Sindbis virus structural polyprotein, which results in the formation of fluorescent capsid-like structures in the absence of viral genomes that associate with the plasma membrane of cells. Mutation of the capsid autoprotease active site blocks structural polyprotein processing and alters the subcellular distribution of capsid fluorescence. Incorporating mCherry into the extracellular domain of the E2 glycoprotein allows the visualization of E2 glycoprotein localization and showed a close association of the E2 and capsid proteins at the plasma membrane as expected. These results suggest that this system is a useful new tool to study alphavirus assembly in live cells and may be useful in identifying molecules that inhibit alphavirus virion formation. PMID:26122073

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome of Turnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta▿

PubMed Central

Beauchemin, Chantal; Boutet, Nathalie; Laliberté, Jean-François

2007-01-01

The RNA genome of Turnip mosaic virus is covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication. PMID:17079311

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

PubMed

Hernández-Sánchez, Itzell E; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P; Jiménez-Bremont, Juan F

2015-01-01

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

PubMed Central

Hernández-Sánchez, Itzell E.; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P.; Jiménez-Bremont, Juan F.

2015-01-01

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization. PMID:26442018

HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling

PubMed Central

Dirk, Brennan S.; Pawlak, Emily N.; Johnson, Aaron L.; Van Nynatten, Logan R.; Jacob, Rajesh A.; Heit, Bryan; Dikeakos, Jimmy D.

2016-01-01

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses. PMID:27841315

Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small Molecule Inhibitors of HIV-1 Nef Dimerization

PubMed Central

Poe, Jerrod A.; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E.

2014-01-01

Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function. PMID:24282155

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

PubMed

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

PubMed Central

Mondal, Samiran; Begum, Nasim A.; Hu, Wenjun; Honjo, Tasuku

2016-01-01

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

In vivo evidence for homo- and heterodimeric interactions of Arabidopsis thaliana dehydrins AtCOR47, AtERD10, and AtRAB18.

PubMed

Hernández-Sánchez, Itzell E; Maruri-López, Israel; Graether, Steffen P; Jiménez-Bremont, Juan F

2017-12-06

Dehydrins (DHNs) are intrinsically disordered proteins that play central roles in plant abiotic stress responses; however, how they work remains unclear. Herein, we report the in planta subcellular localization of Arabidopsis thaliana DHNs AtCOR47, AtERD10, and AtRAB18 through GFP translational fusions. To explore the dimerization ability of the Arabidopsis acidic DHNs AtCOR47 and AtERD10, we conducted an in planta DHN binding assay using the Bimolecular Fluorescence Complementation (BiFC) technique. Our analyses revealed homodimeric interactions for AtCOR47 and AtERD10; interestingly, heterodimeric associations also occurred with these DHNs, and these interactions were observed in the cytosol of tobacco cells. Furthermore, we evaluated whether Arabidopsis basic DHNs, such as AtRAB18, could also interact with itself and/or with AtCOR47 and AtERD10 in the BiFC system. Our data revealed homodimeric RAB18 complexes in the nucleus and cytosol, while heterodimeric associations between AtRAB18 and acidic DHNs occurred only in the cytosol. Finally, we demonstrated the presence of heterodimeric complexes among Arabidopsis AtCOR47, AtERD10, and AtRAB18 DHNs with their acidic ortholog the OpsDHN1 from Opuntia streptacantha; these heterodimeric interactions showed different subcellular distributions. Our results guide DHN research toward a new scenario where DHN/DHN oligomerization could be explored as a part of their molecular mechanism.

Xanthomonas oryzae pv. oryzae TALE proteins recruit OsTFIIAγ1 to compensate for the absence of OsTFIIAγ5 in bacterial blight in rice.

PubMed

Ma, Wenxiu; Zou, Lifang; Ji, Zhiyuan; Xu, Xiameng; Xu, Zhengyin; Yang, Yangyang; Alfano, James R; Chen, Gongyou

2018-04-28

Xanthomonas oryzae pv. oryzae (Xoo), causal agent of bacterial blight (BB) of rice, uses transcription activator-like effectors (TALEs) to interact with the basal transcription factor gama subunit OsTFIIAγ5 (Xa5) and activates transcription of host genes. However, how OsTFIIAγ1, the other OsTFIIAγ protein, functions in the presence of TALEs remains unclear. In this study, we show that OsTFIIAγ1 plays a compensatory role in the absence of Xa5. The expression of OsTFIIAγ1, which is activated by TALE PthXo7, increased the expression of host genes targeted by avirulent and virulent TALEs. Defective OsTFIIAγ1 rice lines showed reduced expression of the TALE-targeted susceptibility (S) genes, OsSWEET11 and OsSWEET14, which resulted in increased BB resistance. Selected TALEs (PthXo1, AvrXa7, and AvrXa27) were evaluated for interactions with OsTFIIAγ1, Xa5 and xa5 (naturally-occurring mutant form of Xa5) using biomolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST). BiFC and MST demonstrated that the three TALEs bind Xa5 and OsTFIIAγ1 with a stronger affinity than xa5. These results provide insight into the complex roles of OsTFIIAγ1 and OsTFIIAγ5 in TALE-mediated host gene transcription. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.

PubMed

Henne, Melina; König, Nicolas; Triulzi, Tiziana; Baroni, Sara; Forlani, Fabio; Scheibe, Renate; Papenbrock, Jutta

2015-01-01

Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vitro, but the physiological roles of these enzymes are not well defined. Several studies in different organisms demonstrate protein-protein interactions of Strs with members of the Trx family. We are interested in investigating the specificity of the interaction between Str and Trx isoforms. In order to use the bimolecular fluorescence complementation (BiFC), several Str and Trx sequences from Arabidopsis thaliana were cloned into the pUC-SPYNE and pUC-SPYCE split-YFP vectors, respectively. Each couple of plasmids containing the sequences for the putative interaction partners were transformed into Arabidopsis protoplasts and screened using a confocal laser scanning microscope. Compartment- and partner-specific interactions could be observed in transformed protoplasts. Replacement of cysteine residues in the redox-active site of Trxs abolished the interaction signal. Therefore, the redox site is not only involved in the redox reaction but also responsible for the interaction with partner proteins. Biochemical assays support a specific interaction among Strs and certain Trxs. Based on the results obtained, the interaction of Strs and Trxs indicates a role of Strs in the maintenance of the cellular redox homeostasis.

Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

PubMed Central

Andersen, Tonni Grube; Nintemann, Sebastian J.; Marek, Magdalena; Halkier, Barbara A.; Schulz, Alexander; Burow, Meike

2016-01-01

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality. PMID:27282591

Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the thrombopoietin receptor (Mpl).

PubMed

Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

2012-01-01

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

Live-Cell Visualization of Intracellular Interaction between a Nuclear Migration Protein (hNUDC) and the Thrombopoietin Receptor (Mpl)

PubMed Central

Zheng, Yuan-Bin; Xiao, Ying-Ying; Tan, Peng; Zhang, Qing; Xu, Peilin

2012-01-01

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway. PMID:23284788

Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3.

PubMed

Li, Shao-Jia; Yin, Xue-Ren; Wang, Wen-Li; Liu, Xiao-Fen; Zhang, Bo; Chen, Kun-Song

2017-06-15

Citric acid is the predominant organic acid of citrus fruit. Degradation of citric acid occurs during fruit development, influencing fruit acidity. Associations of CitAco3 transcripts and citric acid degradation have been reported for citrus fruit. Here, transient overexpression of CitAco3 significantly reduced the citric acid content of citrus leaves and fruits. Using dual luciferase assays, it was shown that CitNAC62 and CitWRKY1 could transactivate the promoter of CitAco3. Subcellular localization results showed that CitWRKY1 was located in the nucleus and CitNAC62 was not. Yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays indicated that the two differently located transcription factors could interact with each other. Furthermore, BiFC showed that the protein-protein interaction occurred only in the nucleus, indicating the potential mobility of CitNAC62 in plant cells. A synergistic effect on citrate content was observed between CitNAC62 and CitWRKY1. Transient overexpression of CitNAC62 or CitWRKY1 led to significantly lower citrate content in citrus fruit. The combined expression of CitNAC62 and CitWRKY1 resulted in lower citrate content compared with the expression of CitNAC62 or CitWRKY1 alone. The transcript abundance of CitAco3 was consistent with the citrate content. Thus, we propose that a complex of CitWRKY1 and CitNAC62 contributes to citric acid degradation in citrus fruit, potentially via modulation of CitAco3. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex.

PubMed

Kaufholdt, David; Baillie, Christin-Kirsty; Meinen, Rieke; Mendel, Ralf R; Hänsch, Robert

2017-01-01

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo . Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnx1, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnx1 as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnx1. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.

GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.

PubMed

Huang, Hung-Hsiang; Hassinen, Antti; Sundaram, Subha; Spiess, Andrej-Nikolai; Kellokumpu, Sakari; Stanley, Pamela

2015-09-15

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

Analyses of pea necrotic yellow dwarf virus-encoded proteins.

PubMed

Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

2017-06-01

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

Mapping transcription factor interactome networks using HaloTag protein arrays.

PubMed

Yazaki, Junshi; Galli, Mary; Kim, Alice Y; Nito, Kazumasa; Aleman, Fernando; Chang, Katherine N; Carvunis, Anne-Ruxandra; Quan, Rosa; Nguyen, Hien; Song, Liang; Alvarez, José M; Huang, Shao-Shan Carol; Chen, Huaming; Ramachandran, Niroshan; Altmann, Stefan; Gutiérrez, Rodrigo A; Hill, David E; Schroeder, Julian I; Chory, Joanne; LaBaer, Joshua; Vidal, Marc; Braun, Pascal; Ecker, Joseph R

2016-07-19

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

PubMed

Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

2015-11-01

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans

PubMed Central

Pandey, Pratima; Bhardwaj, Ashwani; Babu, Kavita

2017-01-01

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the β-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1. PMID:28515212

The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

PubMed

Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

2011-04-01

The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

PubMed Central

Dirk, Brennan S.; Van Nynatten, Logan R.; Dikeakos, Jimmy D.

2016-01-01

Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle. PMID:27775563

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity*

PubMed Central

Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

2014-01-01

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief. PMID:24808184

Targeting the transient receptor potential vanilloid type 1 (TRPV1) assembly domain attenuates inflammation-induced hypersensitivity.

PubMed

Flynn, Robyn; Chapman, Kevin; Iftinca, Mircea; Aboushousha, Reem; Varela, Diego; Altier, Christophe

2014-06-13

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the PBC Class Cofactors

PubMed Central

Hudry, Bruno; Remacle, Sophie; Delfini, Marie-Claire; Rezsohazy, René; Graba, Yacine; Merabet, Samir

2012-01-01

Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes. PMID:22745600

Cell-to-cell Transmission of Polyglutamine Aggregates in C. elegans

PubMed Central

Kim, Dong-Kyu; Cho, Kyu-Won; Ahn, Woo Jung; Perez-Acuña, Dayana; Jeong, Hyunsu; Lee, He-Jin

2017-01-01

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an in vivo model for studying transmission of polyQ aggregates in a highly quantitative manner in real time. HTT exon 1 with expanded polyQ was fused with either N-terminal or C-terminal fragments of Venus fluorescence protein and expressed in pharyngeal muscles and associated neurons, respectively, of C. elegans. Transmission of polyQ proteins was detected using bimolecular fluorescence complementation (BiFC). Mutant polyQ (Q97) was transmitted much more efficiently than wild type polyQ (Q25) and forms numerous inclusion bodies as well. The transmission of Q97 was gradually increased with aging of animal. The animals with polyQ transmission exhibited degenerative phenotypes, such as nerve degeneration, impaired pharyngeal pumping behavior, and reduced life span. The C. elegans model presented here would be a useful in vivo model system for the study of polyQ aggregate propagation and might be applied to the screening of genetic and chemical modifiers of the propagation. PMID:29302199

Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1.

PubMed

Martins, Viviana; Bassil, Elias; Hanana, Mohsen; Blumwald, Eduardo; Gerós, Hernâni

2014-07-01

The Vitis vinifera copper transporter 1 is capable of self-interaction and mediates intracellular copper transport. An understanding of copper homeostasis in grapevine (Vitis vinifera L.) is particularly relevant to viticulture in which copper-based fungicides are intensively used. In the present study, the Vitis vinifera copper transporter 1 (VvCTr1), belonging to the Ctr family of copper transporters, was cloned and functionally characterized. Amino acid sequence analysis showed that VvCTr1 monomers are small peptides composed of 148 amino acids with 3 transmembrane domains and several amino acid residues typical of Ctr transporters. Bimolecular fluorescence complementation (BiFC) demonstrated that Ctr monomers are self-interacting and subcellular localization studies revealed that VvCTr1 is mobilized via the trans-Golgi network, through the pre-vacuolar compartment and located to the vacuolar membrane. The heterologous expression of VvCTr1 in a yeast strain lacking all Ctr transporters fully rescued the phenotype, while a deficient complementation was observed in a strain lacking only plasma membrane-bound Ctrs. Given the common subcellular localization of VvCTr1 and AtCOPT5 and the highest amino acid sequence similarity in comparison to the remaining AtCOPT proteins, Arabidopsis copt5 plants were stably transformed with VvCTr1. The impairment in root growth observed in copt5 seedlings in copper-deficient conditions was fully rescued by VvCTr1, further supporting its involvement in intracellular copper transport. Expression studies in V. vinifera showed that VvCTr1 is mostly expressed in the root system, but transcripts were also present in leaves and stems. The functional characterization of VvCTr-mediated copper transport provides the first step towards understanding the physiological and molecular responses of grapevines to copper-based fungicides.

Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

PubMed Central

Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

2015-01-01

The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

Chitin receptor CERK1 links salt stress and chitin-triggered innate immunity in Arabidopsis.

PubMed

Espinoza, Catherine; Liang, Yan; Stacey, Gary

2017-03-01

In nature, plants need to respond to multiple environmental stresses that require the involvement and fine-tuning of different stress signaling pathways. Cross-tolerance, in which plants pre-treated with chitin (a fungal microbe-associated molecular pattern) have improved salt tolerance, was observed in Arabidopsis, but is not well understood. Here, we show a unique link between chitin and salt signaling mediated by the chitin receptor CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1). Transcriptome analysis revealed that salt stress-induced genes are highly correlated with chitin-induced genes, although this was not observed with other microbe-associated molecular patterns (MAMPs) or with other abiotic stresses. The cerk1 mutant was more susceptible to NaCl than was the wild type. cerk1 plants had an irregular increase of cytosolic calcium ([Ca 2+ ] cyt ) after NaCl treatment. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation experiments indicated that CERK1 physically interacts with ANNEXIN 1 (ANN1), which was reported to form a calcium-permeable channel that contributes to the NaCl-induced [Ca 2+ ] cyt signal. In turn, ann1 mutants showed elevated chitin-induced rapid responses. In short, molecular components previously shown to function in chitin or salt signaling physically interact and intimately link the downstream responses to fungal attack and salt stress. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Visualization of the Drosophila dKeap1-CncC interaction on chromatin illumines cooperative, xenobiotic-specific gene activation

PubMed Central

Deng, Huai; Kerppola, Tom K.

2014-01-01

Interactions among transcription factors control their physiological functions by regulating their binding specificities and transcriptional activities. We implement a strategy to visualize directly the genomic loci that are bound by multi-protein complexes in single cells in Drosophila. This method is based on bimolecular fluorescence complementation (BiFC) analysis of protein interactions on polytene chromosomes. Drosophila Keap1 (dKeap1)-CncC complexes localized to the nucleus and bound chromatin loci that were not bound preferentially by dKeap1 or CncC when they were expressed separately. dKeap1 and CncC binding at these loci was enhanced by phenobarbital, but not by tert-butylhydroquinone (tBHQ) or paraquat. Endogenous dKeap1 and CncC activated transcription of the Jheh (Jheh1, Jheh2, Jheh3) and dKeap1 genes at these loci, whereas CncC alone activated other xenobiotic response genes. Ectopic dKeap1 expression increased CncC binding at the Jheh and dKeap1 gene loci and activated their transcription, whereas dKeap1 inhibited CncC binding at other xenobiotic response gene loci and suppressed their transcription. The combinatorial chromatin-binding specificities and transcriptional activities of dKeap1-CncC complexes mediated the selective activation of different sets of genes by different xenobiotic compounds, in part through feed-forward activation of dKeap1 transcription. PMID:25063457

UNC-1 regulates gap junctions important to locomotion in C. elegans.

PubMed

Chen, Bojun; Liu, Qiang; Ge, Qian; Xie, Jia; Wang, Zhao-Wen

2007-08-07

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

A Small Secreted Virulence-Related Protein Is Essential for the Necrotrophic Interactions of Sclerotinia sclerotiorum with Its Host Plants

PubMed Central

Lyu, Xueliang; Shen, Cuicui; Fu, Yanping; Xie, Jiatao; Jiang, Daohong; Li, Guoqing; Cheng, Jiasen

2016-01-01

Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction. PMID:26828434

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Interactions between mitoNEET and NAF-1 in cells

PubMed Central

Tamir, Sagi; Luo, Yuting; Bai, Fang; Adenwalla, Ammar; Darash-Yahana, Merav; Sohn, Yang-Sung; Jennings, Patricia A.; Azad, Rajeev K.; Onuchic, Jose' N.; Morcos, Faruck; Nechushtai, Rachel

2017-01-01

The NEET proteins mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are required for cancer cell proliferation and resistance to oxidative stress. NAF-1 and mNT are also implicated in a number of other human pathologies including diabetes, neurodegeneration and cardiovascular disease, as well as in development, differentiation and aging. Previous studies suggested that mNT and NAF-1 could function in the same pathway in mammalian cells, preventing the over-accumulation of iron and reactive oxygen species (ROS) in mitochondria. Nevertheless, it is unknown whether these two proteins directly interact in cells, and how they mediate their function. Here we demonstrate, using yeast two-hybrid, in vivo bimolecular fluorescence complementation (BiFC), direct coupling analysis (DCA), RNA-sequencing, ROS and iron imaging, and single and double shRNA lines with suppressed mNT, NAF-1 and mNT/NAF-1 expression, that mNT and NAF-1 directly interact in mammalian cells and could function in the same cellular pathway. We further show using an in vitro cluster transfer assay that mNT can transfer its clusters to NAF-1. Our study highlights the possibility that mNT and NAF-1 function as part of an iron-sulfur (2Fe-2S) cluster relay to maintain the levels of iron and Fe-S clusters under control in the mitochondria of mammalian cells, thereby preventing the activation of apoptosis and/or autophagy and supporting cellular proliferation. PMID:28426722

The Banana Fruit SINA Ubiquitin Ligase MaSINA1 Regulates the Stability of MaICE1 to be Negatively Involved in Cold Stress Response.

PubMed

Fan, Zhong-Qi; Chen, Jian-Ye; Kuang, Jian-Fei; Lu, Wang-Jin; Shan, Wei

2017-01-01

The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis . Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi- in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

Melatonin biosynthesis enzymes recruit WRKY transcription factors to regulate melatonin accumulation and transcriptional activity on W-box in cassava.

PubMed

Wei, Yunxie; Liu, Guoyin; Chang, Yanli; Lin, Daozhe; Reiter, Russel J; He, Chaozu; Shi, Haitao

2018-03-12

Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)-interacting proteins, N-acetylserotonin O-methyltransferase 2 (MeASMT2) interacting proteins, and N-acetylserotonin O-methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two-hybrid, 14 MeTDC2-interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3-interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two-hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co-overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W-box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling.

PubMed

Bueso, Eduardo; Rodriguez, Lesia; Lorenzo-Orts, Laura; Gonzalez-Guzman, Miguel; Sayas, Enric; Muñoz-Bertomeu, Jesús; Ibañez, Carla; Serrano, Ramón; Rodriguez, Pedro L

2014-12-01

Membrane-delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single-subunit RING-type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C-terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1-PYL4 and RSL1-PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half-life, protein interactions or trafficking. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

The Rice AAA-ATPase OsFIGNL1 Is Essential for Male Meiosis

PubMed Central

Zhang, Peipei; Zhang, Yingxin; Sun, Lianping; Sinumporn, Sittipun; Yang, Zhengfu; Sun, Bin; Xuan, Dandan; Li, Zihe; Yu, Ping; Wu, Weixun; Wang, Kejian; Cao, Liyong; Cheng, Shihua

2017-01-01

Meiosis is crucial in reproduction of plants and ensuring genetic diversity. Although several genes involved in homologous recombination and DNA repair have been reported, their functions in rice (Oryza sativa) male meiosis remain poorly understood. Here, we isolated and characterized the rice OsFIGNL1 (OsFidgetin-like 1) gene, encoding a conserved AAA-ATPase, and explored its function and importance in male meiosis and pollen formation. The rice Osfignl1 mutant exhibited normal vegetative growth, but failed to produce seeds and displayed pollen abortion phenotype. Phenotypic comparisons between the wild-type and Osfignl1 mutant demonstrated that OsFIGNL1 is required for anther development, and that the recessive mutation of this gene causes male sterility in rice. Complementation and CRISPR/Cas9 experiments demonstrated that wild-type OsFIGNL1 is responsible for the male sterility phenotype. Subcellular localization showed that OsFIGNL1-green fluorescent protein was exclusively localized in the nucleus of rice protoplasts. Male meiosis in the Osfignl1 mutant exhibited abnormal chromosome behavior, including chromosome bridges and multivalent chromosomes at diakinesis, lagging chromosomes, and chromosome fragments during meiosis. Yeast two-hybrid assays demonstrated OsFIGNL1 could interact with RAD51A1, RAD51A2, DMC1A, DMC1B, and these physical interactions were further confirmed by BiFC assay. Taken together, our results suggest that OsFIGNL1 plays an important role in regulation of male meiosis and anther development. PMID:29021797

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain.

PubMed

Valsecchi, Isabel; Guittard-Crilat, Emilie; Maldiney, Régis; Habricot, Yvette; Lignon, Sabrina; Lebrun, Régine; Miginiac, Emile; Ruelland, Eric; Jeannette, Emmanuelle; Lebreton, Sandrine

2013-09-01

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

PubMed

Wang, Jie; Yao, Wenkong; Wang, Lei; Ma, Fuli; Tong, Weihuo; Wang, Chen; Bao, Rui; Jiang, Changyue; Yang, Yazhou; Zhang, Jianxia; Xu, Yan; Wang, Xiping; Zhang, Chaohong; Wang, Yuejin

2017-10-01

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H 2 O 2 ) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCF VpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H 2 O 2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system. Copyright © 2017 Elsevier B.V. All rights reserved.

Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

PubMed Central

Matsuda-Lennikov, Mami; Suizu, Futoshi; Hirata, Noriyuki; Hashimoto, Manabu; Kimura, Kohki; Nagamine, Tadashi; Fujioka, Yoichiro; Ohba, Yusuke; Iwanaga, Toshihiko; Noguchi, Masayuki

2014-01-01

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P. PMID:24416124

A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK61

PubMed Central

2017-01-01

Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca2+ signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named “SlRd2”. SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2 treatment. Finally, transient expression of SlCipk6 in transgenic N. benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation. PMID:27899535

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

PubMed Central

Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

2014-01-01

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

A comparative approach expands the protein-protein interaction node of the immune receptor XA21 in wheat and rice

PubMed Central

Yang, Baoju; Ruan, Randy; Cantu, Dario; Wang, Xiaodong; Ji, Wanquan; Ronald, Pamela C; Dubcovsky, Jorge

2016-01-01

The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a non-syntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation (BiFC) assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that inter-specific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice. PMID:23957671

The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions

PubMed Central

Xu, Weihui; Meng, Yan; Surana, Priyanka; Fuerst, Greg; Nettleton, Dan; Wise, Roger P.

2015-01-01

Plants have evolved complex regulatory mechanisms to control a multi-layered defense response to microbial attack. Both temporal and spatial gene expression are tightly regulated in response to pathogen ingress, modulating both positive and negative control of defense. BLUFENSINs, small knottin-like peptides in barley, wheat, and rice, are highly induced by attack from fungal pathogens, in particular, the obligate biotrophic fungus, Blumeria graminis f. sp. hordei (Bgh), causal agent of barley powdery mildew. Previous research indicated that Blufensin1 (Bln1) functions as a negative regulator of basal defense mechanisms. In the current report, we show that BLN1 and BLN2 can both be secreted to the apoplast and Barley stripe mosaic virus (BSMV)-mediated overexpression of Bln2 increases susceptibility of barley to Bgh. Bimolecular fluorescence complementation (BiFC) assays signify that BLN1 and BLN2 can interact with each other, and with calmodulin. We then used BSMV-induced gene silencing to knock down Bln1, followed by Barley1 GeneChip transcriptome analysis, to identify additional host genes influenced by Bln1. Analysis of differential expression revealed a gene set enriched for those encoding proteins annotated to nuclear import and the secretory pathway, particularly Importin α1-b and Sec61 γ subunits. Further functional analysis of these two affected genes showed that when silenced, they also reduced susceptibility to Bgh. Taken together, we postulate that Bln1 is co-opted by Bgh to facilitate transport of disease-related host proteins or effectors, influencing the establishment of Bgh compatibility on its barley host. PMID:26089830

Molecular cloning and functional analysis of a blue light receptor gene MdCRY2 from apple (Malus domestica).

PubMed

Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Rui-Fen; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

2013-04-01

MdCRY2 was isolated from apple fruit skin, and its function was analyzed in MdCRY2 transgenic Arabidopsis. The interaction between MdCRY2 and AtCOP1 was found by yeast two-hybrid and BiFC assays. Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development. Investigations of the structure and functions of cryptochromes in plants have largely focused on Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), pea (Pisum sativum), and rice (Oryza sativa). However, no data on the function of CRY2 are available in woody plants. In this study, we isolated a cryptochrome gene, MdCRY2, from apple (Malus domestica). The deduced amino acid sequences of MdCRY2 contain the conserved N-terminal photolyase-related domain and the flavin adenine dinucleotide (FAD) binding domain, as well as the C-terminal DQXVP-acidic-STAES (DAS) domain. Relationship analysis indicates that MdCRY2 shows the highest similarity to the strawberry FvCRY protein. The expression of MdCRY2 is induced by blue/UV-A light, which represents a 48-h circadian rhythm. To investigate the function of MdCRY2, we overexpressed the MdCRY2 gene in a cry2 mutant and wild type (WT) Arabidopsis, assessed the phenotypes of the resulting transgenic plants, and found that MdCRY2 functions to regulate hypocotyl elongation, root growth, flower initiation, and anthocyanin accumulation. Furthermore, we examined the interaction between MdCRY2 and AtCOP1 using a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. These data provide functional evidence for a role of blue/UV-A light-induced MdCRY2 in controlling photomorphogenesis in apple.

A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK6.

PubMed

Gutiérrez-Beltrán, Emilio; Personat, José María; de la Torre, Fernando; Del Pozo, Olga

2017-01-01

Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca 2+ signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named "SlRd2". SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H 2 O 2 treatment. Finally, transient expression of SlCipk6 in transgenic N benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation. © 2017 American Society of Plant Biologists. All Rights Reserved.

Protein Interaction Analysis Provides a Map of the Spatial and Temporal Organization of the Ciliary Gating Zone.

PubMed

Takao, Daisuke; Wang, Liang; Boss, Allison; Verhey, Kristen J

2017-08-07

The motility and signaling functions of the primary cilium require a unique protein and lipid composition that is determined by gating mechanisms localized at the base of the cilium. Several protein complexes localize to the gating zone and may regulate ciliary protein composition; however, the mechanisms of ciliary gating and the dynamics of the gating components are largely unknown. Here, we used the BiFC (bimolecular fluorescence complementation) assay and report for the first time on the protein-protein interactions that occur between ciliary gating components and transiting cargoes during ciliary entry. We find that the nucleoporin Nup62 and the C termini of the nephronophthisis (NPHP) proteins NPHP4 and NPHP5 interact with the axoneme-associated kinesin-2 motor KIF17 and thus spatially map to the inner region of the ciliary gating zone. Nup62 and NPHP4 exhibit rapid turnover at the transition zone and thus define dynamic components of the gate. We find that B9D1, AHI1, and the N termini of NPHP4 and NPHP5 interact with the transmembrane protein SSTR3 and thus spatially map to the outer region of the ciliary gating zone. B9D1, AHI1, and NPHP5 exhibit little to no turnover at the transition zone and thus define components of a stable gating structure. These data provide the first comprehensive map of the molecular orientations of gating zone components along the inner-to-outer axis of the ciliary gating zone. These results advance our understanding of the functional roles of gating zone components in regulating ciliary protein composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wheat TaNPSN SNARE homologues are involved in vesicle-mediated resistance to stripe rust (Puccinia striiformis f. sp. tritici)

PubMed Central

Wang, Xiaodong; Wang, Xiaojie; Deng, Lin; Chang, Haitao; Dubcovsky, Jorge; Feng, Hao; Han, Qingmei; Huang, Lili; Kang, Zhensheng

2014-01-01

Subcellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complexes are critical for determining the specificity of vesicle fusion. NPSN11, a Novel Plant SNARE (NPSN) gene, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant–pathogen interactions are largely unknown. In this study, we cloned and characterized three NPSN genes (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant defence-related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12). TaSYP132 showed a highly specific interaction with TaNPSN11 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132 were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici (Pst). In virus-induced gene silencing (VIGS) assays, resistance of wheat (Triticum aestivum) cultivar Xingzi9104 to the Pst avirulent race CYR23 was reduced by knocking down TaNPSN11, TaNPSN13 and TaSYP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11 or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11 in vesicle-mediated resistance to stripe rust. PMID:24963004

Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models.

PubMed

Brasil, Aline A; Magalhães, Rayne S S; De Carvalho, Mariana D C; Paiva, Isabel; Gerhardt, Ellen; Pereira, Marcos D; Outeiro, Tiago F; Eleutherio, Elis C A

2018-06-01

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

Mapping regions of Epstein-Barr virus (EBV) glycoprotein B (gB) important for fusion function with gH/gL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plate, Aileen E.; Reimer, Jessica J.; Jardetzky, Theodore S.

Glycoproteins gB and gH/gL are required for entry of Epstein-Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gBmore » with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB-gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.« less

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2014-04-01

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2011-06-07

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2015-07-14

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

PubMed

Li, Xiao-Dan; Deng, Cheng-Lin; Ye, Han-Qing; Zhang, Hong-Lei; Zhang, Qiu-Yan; Chen, Dong-Dong; Zhang, Pan-Tao; Shi, Pei-Yong; Yuan, Zhi-Ming; Zhang, Bo

2016-06-15

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Reliability and quality of water isotope data collected with a low-budget rain collector.

PubMed

Prechsl, Ulrich E; Gilgen, Anna K; Kahmen, Ansgar; Buchmann, Nina

2014-04-30

Low-budget rain collectors for water isotope analysis, such as the 'ball-in-funnel type collector' (BiFC), are widely used in studies on stable water isotopes of rain. To date, however, an experimental quality assessment of such devices in relation to climatic factors does not exist. We used Cavity Ring-Down Spectrometry (CRDS) to quantify the effects of evaporation on the δ(18)O values of reference water under controlled conditions as a function of the elapsed time between rainfall and collection for isotope analysis, the sample volume and the relative humidity (RH: 31% and 67%; 25 °C). The climate chamber conditions were chosen to reflect the warm and dry end of field conditions that favor evaporative enrichment (EE). We also tested the performance of the BiFC in the field, and compared our δ(2)H/δ(18)O data obtained by isotope ratio mass spectrometry (IRMS) with those from the Swiss National Network for the Observation of Isotopes in the Water Cycle (ISOT). The EE increased with time, with a 1‰ increase in the δ(18)O values after 10 days (RH: 25%; 25 °C; 35 mL (corresponding to a 5 mm rain event); p <0.001). The sample volume strongly affected the EE (max. value +1.5‰ for 7 mL samples (i.e., 1 mm rain events) after 72 h at 31% and 67% RH; p <0.001), whereas the relative humidity had no significant effect. Using the BiFC in the field, we obtained very tight relationships of the δ(2)H/δ(18)O values (r(2) ≥ 0.95) for three sites along an elevational gradient, not significantly different from that of the next ISOT station. Since the chosen experimental conditions were extreme compared with the field conditions, it was concluded that the BiFC is a highly reliable and inexpensive collector of rainwater for isotope analysis. Copyright © 2014 John Wiley & Sons, Ltd.

PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells.

PubMed

Lee, Dong Kun; Park, Jung Yeon; Yoo, Jae Cheal; Byun, Eun Hye; Bae, Yeon-Ju; Lee, Young-Sun; Park, Nammi; Kang, Dawon; Han, Jaehee; Park, Jae Yong; Hwang, Eunmi; Hong, Seong-Geun

2018-06-21

Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

PubMed Central

Szczesny, Roman J.; Kowalska, Katarzyna; Klosowska-Kosicka, Kamila; Chlebowski, Aleksander; Owczarek, Ewelina P.; Warkocki, Zbigniew; Kulinski, Tomasz M.; Adamska, Dorota; Affek, Kamila; Jedroszkowiak, Agata; Kotrys, Anna V.; Tomecki, Rafal; Krawczyk, Pawel S.; Borowski, Lukasz S.; Dziembowski, Andrzej

2018-01-01

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq. PMID:29590189

Ovate family protein1 interaction with BLH3 regulates transition timing from vegetative to reproductive phase in Arabidopsis

DOE PAGES

Zhang, Liguo; Zhang, Xiaofei; Ju, Hanxun; ...

2016-01-23

We study the Three-Amino-acid-Loop-Extension(TALE) homeodomain transcription factor BLH3 that regulates timing of transition from vegetative to reproductive phase. Previous preliminary results obtained using large-scale yeast two-hybrids indicate that BLH3 protein possibly interact with Ovate Family Proteins(OFPs) transcription co-regulators. Nevertheless, it is uncertain whether OFP1–BLH3 complex is involved in regulation of timing of transition from vegetative to reproductive phase in Arabidopsis. The interaction between BLH3 and OFP1 was re-tested and verified by a yeast two-hybrid system. We found that the BLH3–OFP1 interaction was mainly mediated through the BLH3 homeodomain. Meanwhile, this interaction was further confirmed by bimolecular fluorescence complementation (BiFC) inmore » vivo. In addition, by establishing protoplast transient expression, we discovered that BLH3 acts as a transcriptional activator, whereas OFP1 functioned as a repressor. The interactions between OFP1 and BLH3 can reduce BLH3 transcriptional activity. The ofp1 mutant lines and blh3 mutant lines, OFP1 overexpress lines and BLH3 overexpress lines can both influence timing of transition from vegetative to reproductive phase. Furthermore, 35s:OFP1/blh3 plants exhibited flowering and leaf quantity similar to that of the wild-type controls. 35s:BLH3/ofp1 plants flowered earlier and had less leaves than wild-type controls, indicating that OFP1 protein might depend partially on BLH3 in its function to regulate the timing of transition from vegetative to reproductive phase. In conclusion, these results support our assumption that, by interacting with OFP1, BLH3 forms a functional protein complex that controls timing of progression from vegetative to reproductive phase, and OFP1 might negatively regulate BLH3 or the BLH-KNOX complex, an important interaction for sustaining the normal transition from vegetative to reproductive phase.« less

hfAIM: A reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated Atg8-interacting motifs in various organisms

PubMed Central

Xie, Qingjun; Tzfadia, Oren; Levy, Matan; Weithorn, Efrat; Peled-Zehavi, Hadas; Van Parys, Thomas; Van de Peer, Yves; Galili, Gad

2016-01-01

ABSTRACT Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirements—the presence of acidic amino acids and the absence of positively charged amino acids in certain positions—to reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link: http://bioinformatics.psb.ugent.be/hfAIM/. PMID:27071037

Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites.

PubMed

Lungu, Cristiana; Pinter, Sabine; Broche, Julian; Rathert, Philipp; Jeltsch, Albert

2017-09-21

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.Tools for imaging epigenetic modifications can shed light on the regulation of epigenetic processes. Here, the authors present a fluorescence complementation approach for detection of DNA and histone methylation at endogenous genomic sites allowing following of dynamic changes of these marks by live-cell microscopy.

Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).

PubMed

Liang, Wanwan; Yang, Bo; Yu, Bao-Jun; Zhou, Zili; Li, Cui; Jia, Ming; Sun, Yun; Zhang, Yue; Wu, Feifei; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

2013-06-11

Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses. We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization. We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

PubMed Central

Moores, J C; Magazin, M; Ditta, G S; Leong, J

1984-01-01

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin. Images PMID:6690426

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

PubMed Central

Vélez-Bermúdez, Isabel C.; Carretero-Paulet, Lorenzo; Lumbreras, Victoria; Pagès, Montserrat

2011-01-01

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes. PMID:21789193

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

PubMed

Calderón, Aingeru; Ortiz-Espín, Ana; Iglesias-Fernández, Raquel; Carbonero, Pilar; Pallardó, Federico Vicente; Sevilla, Francisca; Jiménez, Ana

2017-04-01

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Conservation of Chitin-Induced MAPK Signaling Pathways in Rice and Arabidopsis.

PubMed

Yamada, Kenta; Yamaguchi, Koji; Yoshimura, Satomi; Terauchi, Akira; Kawasaki, Tsutomu

2017-06-01

Perception of microbe-associated molecular patterns (MAMPs) including chitin by pattern recognition receptors (PRRs) rapidly induces activation of mitogen-activated protein kinase (MAPK) cascades. However, how PRRs transmit immune signals to the MAPK cascade is largely unknown. Recently, Arabidopsis receptor-like cytoplasmic kinase PBL27 has been reported to activate MAPKs through phosphorylation of AtMAPKKK5 in the chitin signaling pathway. In this study, we found that OsRLCK185, a rice ortholog of PBL27, regulates chitin-induced MAPK activation in a similar fashion to PBL27 in rice. Upon chitin perception, OsRLCK185 is phosphorylated by OsCERK1, a component of the chitin receptor complex. OsRLCK185 interacted with OsMAPKKK11 and OsMAPKKK18, rice orthologs of AtMAPKKK5, in yeast two-hybrid assays. Silencing of both OsMAPKKK11 and OsMAPKKK18 significantly reduced chitin-induced activation of OsMPK3 and OsMPK6. Expression levels of OsMAPKKK18 were much higher than that of OsMAPKKK11 in rice cells, which was consistent with the fact that the Osmapkkk11 single mutation did not affect MAPK activation. This result suggested that OsMAPKKK18 plays a more important role than OsMAPKKK11 in the chitin-induced activation of OsMPK3 and OsMPK6. The bimolecular fluorescence complementation (BiFC) experiment indicated that OsRLCK185 interacted with OsMAPKKK18 at the plasma membrane in planta. In vitro phosphorylation experiments showed that OsRLCK185 directly phosphorylates OsMAPKKK18. Furthermore, OsMAPKKK18 interacted with the MAPKK OsMKK4, the upstream component of OsMPK3/6. These results suggested that OsRLCK185 connects the chitin receptor to the MAPK cascade consisting of OsMAPKKK18-OsMKK4-OsMPK3/6. Our data revealed that chitin-induced MAPK activation in rice and Arabidopsis is regulated by common homologous elements. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

PubMed

Zhou, Jing; Chen, Jing; Zhang, Xiao-Min; Gao, Zhi-Can; Liu, Chun-Chun; Zhang, Yun-Na; Hou, Jin-Xiu; Li, Zhao-Yao; Kan, Lin; Li, Wen-Liang; Zhou, Bin

2018-04-01

Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S -transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B. IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo , but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities. Copyright © 2018 American Society for Microbiology.

Functional characterization of lysosomal interaction of Akt with VRK2.

PubMed

Hirata, Noriyuki; Suizu, Futoshi; Matsuda-Lennikov, Mami; Tanaka, Tsutomu; Edamura, Tatsuma; Ishigaki, Satoko; Donia, Thoria; Lithanatudom, Pathrapol; Obuse, Chikashi; Iwanaga, Toshihiko; Noguchi, Masayuki

2018-06-05

Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci.

PubMed

Milbredt, Sarah; Waldminghaus, Torsten

2017-06-07

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. Copyright © 2017 Milbredt and Waldminghaus.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

PubMed Central

Milbredt, Sarah; Waldminghaus, Torsten

2017-01-01

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. PMID:28450375

Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

PubMed

Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

2015-02-01

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

Immunofluorescence Staining — EDRN Public Portal

Cancer.gov

Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xiaofei; College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036; Deng, Ping

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings themore » split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.« less

Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation

PubMed Central

Macpherson, Lindsey J.; Zaharieva, Emanuela E.; Kearney, Patrick J.; Alpert, Michael H.; Lin, Tzu-Yang; Turan, Zeynep; Lee, Chi-Hon; Gallio, Marco

2015-01-01

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system. PMID:26635273

Functional characterization of an apple apomixis-related MhFIE gene in reproduction development.

PubMed

Liu, Dan-Dan; Dong, Qing-Long; Sun, Chao; Wang, Qing-Lian; You, Chun-Xiang; Yao, Yu-Xin; Hao, Yu-Jin

2012-04-01

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Comparative analysis of protein-protein interactions in the defense response of rice and wheat.

PubMed

Cantu, Dario; Yang, Baoju; Ruan, Randy; Li, Kun; Menzo, Virginia; Fu, Daolin; Chern, Mawsheng; Ronald, Pamela C; Dubcovsky, Jorge

2013-03-12

Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12). Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages. The observed high conservation of interactions between proteins that serve as key regulators of the rice defense response suggests that the existing rice interactome can be used to predict interactions in wheat. Such predictions are less reliable for nodes that have undergone a different history of duplications and sub-functionalization in the two lineages.

Detection of Heteromers Formed by Cannabinoid CB1, Dopamine D2, and Adenosine A2A G-Protein-Coupled Receptors by Combining Bimolecular Fluorescence Complementation and Bioluminescence Energy Transfer

PubMed Central

Navarro, Gemma; Carriba, Paulina; Gandí, Jorge; Ciruela, Francisco; Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Canela, Enric I.; Lluis, Carmen; Franco, Rafael

2008-01-01

Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells. PMID:18956124

Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

PubMed Central

Liv, Nalan; van Oosten Slingeland, Daan S. B.; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W.; Hoogenboom, Jacob P.

2016-01-01

We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

ICE1 of Pyrus ussuriensis functions in cold tolerance by enhancing PuDREBa transcriptional levels through interacting with PuHHP1

NASA Astrophysics Data System (ADS)

Huang, Xiaosan; Li, Kongqing; Jin, Cong; Zhang, Shaoling

2015-12-01

ICE1 transcription factor plays an important role in plant cold stress via regulating the expression of stress-responsive genes. In this study, a PuICE1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. The expression levels of the PuICE1 were induced by cold, dehydration and salt, with the greatest induction under cold conditions. PuICE1 was localized in the nucleus and could bind specifically to the MYC element in the PuDREBa promoter. The PuICE1 fused to the GAL4 DNA-binding domain to have transcriptional activation activity. Ectopic expression of the PuICE1 in tomato conferred enhanced tolerance to cold stress at cold temperatures, less electrolyte leakage, less MDA content, higher chlorophyll content, higher survival rate, higher proline content, higher activities of enzymes. In additon, steady-state mRNA levels of six stress-responsive genes coding for either functional or regulatory genes were induced to higher levels in the transgenic lines by cold stress. Yeast two-hybrid, transient assay, split luciferase complementation and BiFC assays all revealed that PuHHP1 protein can physically interact with PuICE1. Taken together, these results demonstrated that PuICE1 plays a positive role in cold tolerance, which may be due to enhancement of PuDREBa transcriptional levels through interacting with the PuHHP1.

Fluorescence lifetime assays: current advances and applications in drug discovery.

PubMed

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

X-ray-induced fluorescent centers formation in zinc- phosphate glasses doped with Ag and Cu ions

NASA Astrophysics Data System (ADS)

Klyukin, D. A.; Pshenova, A. S.; Sidorov, A. I.; Stolyarchuk, M. V.

2016-08-01

Fluorescent properties of silver and copper doped zinc-phosphate glasses were studied. By X-ray irradiation of silver and copper co-doped glasses we could create and identify new emission centers which do not exist in single-doped samples. Doping of the glass with both silver and copper ions leads to the increase of quantum yield by 2.7 times. The study was complemented by quantum chemical calculations using the time-dependent density functional theory. It was shown that fluorescence may be attributed to the formation of mixed Ag-Cu molecular clusters.

Immunity to human cytomegalovirus measured and compared by complement fixation, indirect fluorescent-antibody, indirect hemagglutination, and enzyme-linked immunosorbent assays.

PubMed Central

Brandt, J A; Kettering, J D; Lewis, J E

1984-01-01

The complement fixation test is currently the test employed most frequently to determine the presence of antibody to human cytomegalovirus. Several other techniques have been adapted for this purpose. A comparison of cytomegalovirus antibody titers was made between the complement fixation test, a commercially available enzyme-linked immunosorbent assay, an indirect immunofluorescent technique, and a modified indirect hemagglutination test. Forty-three serum samples were tested for antibodies by each of the above procedures. The enzyme-linked immunosorbent, immunofluorescent, and indirect hemagglutination assays were in close agreement on all samples tested; the titers obtained with these methods were all equal to or greater than the complement fixation titer for 38 of the 41 samples (92.6%). Two samples were anticomplementary in the complement fixation test but gave readable results in the other tests. The complement fixation test was the least sensitive of the procedures examined. The commercial enzyme-linked immunosorbent assay system was the most practical method and offered the highest degree of sensitivity in detecting antibodies to cytomegalovirus. PMID:6321544

Characterization of phagocytic hemocytes in Ornithodoros moubata (Acari: Ixodidae).

PubMed

Inoue, N; Hanada, K; Tsuji, N; Igarashi, I; Nagasawa, H; Mikami, T; Fujisaki, K

2001-07-01

Effects of fetal bovine serum (FBS) and complement on phagocytic activity in Ornithodaros moubata (Murray 1877) hemocytes and protease activity in the hemocytes were examined. At least three morphologically different cell types, granulocytes, plasmatocytes, and prohemocytes, were detected in hemolymph of O. moubata, and granulocytes and plasmatocytes showed phagocytic activity. FBS altered phagocytic activity of granulocytes, and complement affected phagocytic activity of plasmatocytes. Ticks were inoculated with fluorescent polystyrene beads in combination with FBS or complement. The average number of beads in granulocytes was significantly higher in the FBS injected group than the control (P < 0.01). The percentage of bead-ingesting plasmatocytes in complement inoculated ticks was significantly lower than that in heat-inactivated complement inoculated and control ticks (P < 0.05). Proteases of tick hemocytes localized in small granules in the cytoplasm not only in phagocytic hemocytes but also in prohemocytes. Results suggested modulation of tick hemocyte function through serum components, and digestion of phagocytosed foreign bodies in the hemocytes.

Protein subcellular localization assays using split fluorescent proteins

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Is the flower fluorescence relevant in biocommunication?

NASA Astrophysics Data System (ADS)

Iriel, Analía; Lagorio, María Gabriela

2010-10-01

Flower fluorescence has been previously proposed as a potential visual signal to attract pollinators. In this work, this point was addressed by quantitatively measuring the fluorescence quantum yield ( Φ f) for flowers of Bellis perennis (white, yellow, pink, and purple), Ornithogalum thyrsoides (petals and ovaries), Limonium sinuatum (white and yellow), Lampranthus productus (yellow), Petunia nyctaginiflora (white), Bougainvillea spectabilis (white and yellow), Antirrhinum majus (white and yellow), Eustoma grandiflorum (white and blue), Citrus aurantium (petals and stigma), and Portulaca grandiflora (yellow). The highest values were obtained for the ovaries of O. thyrsoides ( Φ f = 0.030) and for Citrus aurantium petals ( Φ f = 0.014) and stigma ( Φ f = 0.013). Emitted photons as fluorescence were compared with reflected photons. It was concluded that the fluorescence emission is negligible compared to the reflected light, even for the most fluorescent samples, and it may not be considered as an optical signal in biocommunication. The work was complemented with the calculation of quantum catches for each studied flower species to describe the visual sensitization of eye photoreceptors.

Fluorescence-Guided Surgery

PubMed Central

Nagaya, Tadanobu; Nakamura, Yu A.; Choyke, Peter L.; Kobayashi, Hisataka

2017-01-01

Surgical resection of cancer remains an important treatment modality. Despite advances in preoperative imaging, surgery itself is primarily guided by the surgeon’s ability to locate pathology with conventional white light imaging. Fluorescence-guided surgery (FGS) can be used to define tumor location and margins during the procedure. Intraoperative visualization of tumors may not only allow more complete resections but also improve safety by avoiding unnecessary damage to normal tissue which can also reduce operative time and decrease the need for second-look surgeries. A number of new FGS imaging probes have recently been developed, complementing a small but useful number of existing probes. In this review, we describe current and new fluorescent probes that may assist FGS. PMID:29312886

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

PubMed

Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

2012-01-01

The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

Nuclear Resonance Fluorescence of U-235 above 3 MeV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, Glen A.; Caggiano, Joseph A.; Miller, Erin A.

Pacific Northwest National Laboratory and Passport Systems have collaborated to conduct measurements to search for a nuclear resonance fluorescence response of U-235 from 3 to 5 MeV using an 8 g sample of highly enriched uranium. These new measurements complement previously reported measurements below 3 MeV. Preliminary analysis indicates that no strong resonances exist for U-235 in this energy range. A second set of measurements focused on a signature search in the 5 to 10 MeV range is still under analysis.

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

PubMed

Hashimoto, Junko; Watanabe, Taku; Seki, Tatsuya; Karasawa, Satoshi; Izumikawa, Miho; Seki, Tomoe; Iemura, Shun-Ichiro; Natsume, Tohru; Nomura, Nobuo; Goshima, Naoki; Miyawaki, Atsushi; Takagi, Motoki; Shin-Ya, Kazuo

2009-09-01

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.

In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.

PubMed

Hoff, Kevin G; Culler, Stephanie J; Nguyen, Peter Q; McGuire, Ryan M; Silberg, Jonathan J; Smolke, Christina D

2009-12-24

A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins. Copyright 2009 Elsevier Ltd. All rights reserved.

Chemometric classification of Chinese lager beers according to manufacturer based on data fusion of fluorescence, UV and visible spectroscopies.

PubMed

Tan, Jin; Li, Rong; Jiang, Zi-Tao

2015-10-01

We report an application of data fusion for chemometric classification of 135 canned samples of Chinese lager beers by manufacturer based on the combination of fluorescence, UV and visible spectroscopies. Right-angle synchronous fluorescence spectra (SFS) at three wavelength difference Δλ=30, 60 and 80 nm and visible spectra in the range 380-700 nm of undiluted beers were recorded. UV spectra in the range 240-400 nm of diluted beers were measured. A classification model was built using principal component analysis (PCA) and linear discriminant analysis (LDA). LDA with cross-validation showed that the data fusion could achieve 78.5-86.7% correct classification (sensitivity), while those rates using individual spectroscopies ranged from 42.2% to 70.4%. The results demonstrated that the fluorescence, UV and visible spectroscopies complemented each other, yielding higher synergic effect. Copyright © 2015 Elsevier Ltd. All rights reserved.

Is the flower fluorescence relevant in biocommunication?

PubMed

Iriel, Analía; Lagorio, María Gabriela

2010-10-01

Flower fluorescence has been previously proposed as a potential visual signal to attract pollinators. In this work, this point was addressed by quantitatively measuring the fluorescence quantum yield (Φ(f)) for flowers of Bellis perennis (white, yellow, pink, and purple), Ornithogalum thyrsoides (petals and ovaries), Limonium sinuatum (white and yellow), Lampranthus productus (yellow), Petunia nyctaginiflora (white), Bougainvillea spectabilis (white and yellow), Antirrhinum majus (white and yellow), Eustoma grandiflorum (white and blue), Citrus aurantium (petals and stigma), and Portulaca grandiflora (yellow). The highest values were obtained for the ovaries of O. thyrsoides (Φ(f) = 0.030) and for Citrus aurantium petals (Φ(f) = 0.014) and stigma (Φ(f) = 0.013). Emitted photons as fluorescence were compared with reflected photons. It was concluded that the fluorescence emission is negligible compared to the reflected light, even for the most fluorescent samples, and it may not be considered as an optical signal in biocommunication. The work was complemented with the calculation of quantum catches for each studied flower species to describe the visual sensitization of eye photoreceptors.

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.

2015-08-15

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of themore » P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.« less

Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications

PubMed Central

Ma, Dinglong; Bec, Julien; Yankelevich, Diego R.; Gorpas, Dimitris; Fatakdawala, Hussain; Marcu, Laura

2014-01-01

Abstract. We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures. PMID:24898604

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle.

PubMed

Santos, Silvia; Chu, Kengyeh K; Lim, Daryl; Bozinovic, Nenad; Ford, Tim N; Hourtoule, Claire; Bartoo, Aaron C; Singh, Satish K; Mertz, Jerome

2009-01-01

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle

NASA Astrophysics Data System (ADS)

Santos, Silvia; Chu, Kengyeh K.; Lim, Daryl; Bozinovic, Nenad; Ford, Tim N.; Hourtoule, Claire; Bartoo, Aaron C.; Singh, Satish K.; Mertz, Jerome

2009-05-01

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-28

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer obtained spatially resolved measurements of Ti K-α emission. Density profiles were measured from K-α intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-α spectra to spectra from CRETIN simulations. This work shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes

PubMed Central

Sánchez-Aparicio, M. T.; Ayllón, J.; Leo-Macias, A.; Wolff, T.

2016-01-01

ABSTRACT The retinoic acid-inducible gene 1 (RIG-I) signaling pathway is essential for the recognition of viruses and the initiation of host interferon (IFN)-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds mitochondrial antiviral signaling protein (MAVS), leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway through the use of bimolecular fluorescence complementation (BiFC) and super-resolution microscopy. Dimers of RIG-I, TRIM25, and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25-mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus-infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. In contrast, the influenza A virus NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm and inhibits the formation of TRIM25 homocomplexes but not the formation of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25, and MAVS, in the presence or absence of two viral IFN antagonistic proteins. IMPORTANCE The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response. PMID:27807226

Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes.

PubMed

Sánchez-Aparicio, M T; Ayllón, J; Leo-Macias, A; Wolff, T; García-Sastre, A

2017-01-15

The retinoic acid-inducible gene 1 (RIG-I) signaling pathway is essential for the recognition of viruses and the initiation of host interferon (IFN)-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds mitochondrial antiviral signaling protein (MAVS), leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway through the use of bimolecular fluorescence complementation (BiFC) and super-resolution microscopy. Dimers of RIG-I, TRIM25, and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25-mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus-infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. In contrast, the influenza A virus NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm and inhibits the formation of TRIM25 homocomplexes but not the formation of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25, and MAVS, in the presence or absence of two viral IFN antagonistic proteins. The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response. Copyright © 2017 American Society for Microbiology.

Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

PubMed

Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

2015-05-01

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

[Variations of peripheral blood autoantibody, immunoglobuliln, and complement levels in patients with non-lactational mastitis and their clinical significances].

PubMed

Xu, Rui; Guo, Qian-Qian; Yang, Le-Ping; Lai, Mi-Lin; Tong, Lin

2016-08-20

To detect the variations in peripheral blood levels of autoantibodies, immunoglobulilns and complements in patients with non-lactational mastitis and investigate whether non-lactational mastitis is an autoimmune disease with immune dysfunction. Seven-eight patients with non-lactational mastitis treated in our hospital between September 2013 and May 2015 and 88 healthy women (control) were examined for peripheral blood levels of antinuclear antibody (ANA), anti-histone antibody (AHA), immunoglobulins (IgA, IgM, and IgG) and complements (C3, C4, and total complements). s Of the 78 patients with non-lactational mastitis, 50 (64.10%) were positive of ANA showing mainly the granular and cytoplasmic granular fluorescence patterns, and the positivity rate was significantly higher than that in the control group (P<0.000). Twenty-eight (36.00%) of the patients were positive of AHA, a rate significantly higher than that in the control group (P<0.000). The levels of IgA, IgM, C4, and total complements levels were all significantly elevated in the patients compared with those in the control group (P<0.05). Patients with non-lactational mastitis have abnormal changes in peripheral blood levels of immunoglobulins and complements with high positivity rates for ANA and AHA, indicating that non-lactational mastitis is an autoimmune disease with immune dysfunction.

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells

PubMed Central

Lubbeck, Jennifer L.; Dean, Kevin M.; Ma, Hairong; Palmer, Amy E.; Jimenez, Ralph

2012-01-01

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts, however the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, non-temporally-coincident excitation beams. Here, we characterize the design and operation of a cytometer with a 3-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW – 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multi-channel fluorescence cytometry, and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching. PMID:22424298

Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

PubMed Central

Ichimura, Taro; Jin, Takashi; Fujita, Hideaki; Higuchi, Hideo; Watanabe, Tomonobu M.

2014-01-01

Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. PMID:25120488

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Inverse transport problems in quantitative PAT for molecular imaging

NASA Astrophysics Data System (ADS)

Ren, Kui; Zhang, Rongting; Zhong, Yimin

2015-12-01

Fluorescence photoacoustic tomography (fPAT) is a molecular imaging modality that combines photoacoustic tomography with fluorescence imaging to obtain high-resolution imaging of fluorescence distributions inside heterogeneous media. The objective of this work is to study inverse problems in the quantitative step of fPAT where we intend to reconstruct physical coefficients in a coupled system of radiative transport equations using internal data recovered from ultrasound measurements. We derive uniqueness and stability results on the inverse problems and develop some efficient algorithms for image reconstructions. Numerical simulations based on synthetic data are presented to validate the theoretical analysis. The results we present here complement these in Ren K and Zhao H (2013 SIAM J. Imaging Sci. 6 2024-49) on the same problem but in the diffusive regime.

In vivo protein stabilization based on fragment complementation and a split GFP system.

PubMed

Lindman, Stina; Hernandez-Garcia, Armando; Szczepankiewicz, Olga; Frohm, Birgitta; Linse, Sara

2010-11-16

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Guo; Institute of Neuroscience, Department of Neurobiology, Second Military Medical University, Shanghai 200433; Yang Huayan

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to {beta}{sub 2} subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasmamore » membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.« less

LucY: A Versatile New Fluorescent Reporter Protein

PubMed Central

Auldridge, Michele E.; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, George N.; Mead, David; Steinmetz, Eric J.

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions. PMID:25906065

LucY: A Versatile New Fluorescent Reporter Protein.

PubMed

Auldridge, Michele E; Cao, Hongnan; Sen, Saurabh; Franz, Laura P; Bingman, Craig A; Yennamalli, Ragothaman M; Phillips, George N; Mead, David; Steinmetz, Eric J

2015-01-01

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.

LucY: A versatile new fluorescent reporter protein

DOE PAGES

Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; ...

2015-04-23

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrastmore » to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.« less

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-22

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer was used to obtain spatially-resolved measurements of Ti K-more » $$\\alpha$$ emission. Density profiles were measured from K-$$\\alpha$$ intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-$$\\alpha$$ spectra to spectra from CRETIN simulations. This study shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.« less

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

NASA Astrophysics Data System (ADS)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

Complement factor H: spatial and temporal expression and localization in the eye.

PubMed

Mandal, Md Nawajes A; Ayyagari, Radha

2006-09-01

Complement factor H (CFH) is a component of the mammalian complement system, which regulates the alternative pathway of complement activation and protects the host cell from inappropriate complement activation. CFH is a key regulator of innate immunity, and CFH deficiency leads to membranoproliferative glomerulonephritis type II. A variation in human CFH, Y402H, has been shown to be associated with an increased risk for age-related macular degeneration. The authors describe studies on the spatial and temporal expression of the CFH gene and localization of this protein in ocular tissues to gain insight into its role in the eye. CFH expression in human and mouse tissues was studied by quantitative RT-PCR and Western blot analysis, and localization of CFH was studied by immunohistochemical analysis followed by fluorescence microscopy. In human and mouse, CFH expression was found to be similar to the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye its expression increased with age. A significant level of CFH expression is maintained in different ocular tissues during development and aging. Sustained high levels of CFH expression in eye tissues suggest that this protein may play a role in protecting these tissues from indiscriminate complement activation and inflammatory insult.

Understanding Unimer Exchange Processes in Block Copolymer Micelles using NMR Diffusometry, Time-Resolved NMR, and SANS

NASA Astrophysics Data System (ADS)

Madsen, Louis; Kidd, Bryce; Li, Xiuli; Miller, Katherine; Cooksey, Tyler; Robertson, Megan

Our team seeks to understand dynamic behaviors of block copolymer micelles and their interplay with encapsulated cargo molecules. Quantifying unimer and cargo exchange rates micelles can provide critical information for determining mechanisms of unimer exchange as well as designing systems for specific cargo release dynamics. We are exploring the utility of NMR spectroscopy and diffusometry techniques as complements to existing SANS and fluorescence methods. One promising new method involves time-resolved NMR spin relaxation measurements, wherein mixing of fully protonated and 2H-labeled PEO-b-PCL micelles solutions shows an increase in spin-lattice relaxation time (T1) with time after mixing. This is due to a weakening in magnetic environment surrounding 1H spins as 2H-bearing unimers join fully protonated micelles. We are measuring time constants for unimer exchange of minutes to hours, and we expect to resolve times of <1 min. This method can work on any solution NMR spectrometer and with minimal perturbation to chemical structure (as in dye-labelled fluorescence methods). Multimodal NMR can complement existing characterization tools, expanding and accelerating dynamics measurements for polymer micelle, nanogel, and nanoparticle developers.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2017-03-27

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Multimodal imaging of vascular grafts using time-resolved fluorescence and ultrasound

NASA Astrophysics Data System (ADS)

Fatakdawala, Hussain; Griffiths, Leigh G.; Wong, Maelene L.; Humphrey, Sterling; Marcu, Laura

2015-02-01

The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in vivo. Traditional methods for the same are destructive, inefficient in time and cost and do not allow time-lapse measurements from the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular (hyperplasia) content via changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by structural changes observed in UBM pertaining to graft integration and intimal thickness over the grafted region. These results encourage the future application of a catheter-based technique that combines these imaging modalities for non-destructive characterization of vascular grafts in vivo.

A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

PubMed

Ning, Dianhua; He, Changtian; Liu, Zhengjie; Liu, Cui; Wu, Qilong; Zhao, TingTing; Liu, Renyong

2017-05-21

Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.

Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

PubMed

Thong, Patricia S P; Tandjung, Stephanus S; Movania, Muhammad Mobeen; Chiew, Wei-Ming; Olivo, Malini; Bhuvaneswari, Ramaswamy; Seah, Hock-Soon; Lin, Feng; Qian, Kemao; Soo, Khee-Chee

2012-05-01

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

Bimolecular fluorescence complementation studies support an in vivo interaction between the F-BOX protein COLD TEMPERATURE GERMINATING10 and PHYTOCHROME INTERACTING FACTOR1

USDA-ARS?s Scientific Manuscript database

The Arabidopsis thaliana F-BOX protein COLD TEMPERATURE GERMINATING10 (CTG10) was identified from an activation tagged mutant screen as causing seeds to complete germination faster than wild type at 10°C when its expression is increased (Salaita et al. 2005. J. Exp. Bot. 56: 2059). Our unpublished d...

Ultrasound guided fluorescence molecular tomography with improved quantification by an attenuation compensated born-normalization and in vivo preclinical study of cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Baoqiang; Berti, Romain; Abran, Maxime

2014-05-15

Ultrasound imaging, having the advantages of low-cost and non-invasiveness over MRI and X-ray CT, was reported by several studies as an adequate complement to fluorescence molecular tomography with the perspective of improving localization and quantification of fluorescent molecular targets in vivo. Based on the previous work, an improved dual-modality Fluorescence-Ultrasound imaging system was developed and then validated in imaging study with preclinical tumor model. Ultrasound imaging and a profilometer were used to obtain the anatomical prior information and 3D surface, separately, to precisely extract the tissue boundary on both sides of sample in order to achieve improved fluorescence reconstruction. Furthermore,more » a pattern-based fluorescence reconstruction on the detection side was incorporated to enable dimensional reduction of the dataset while keeping the useful information for reconstruction. Due to its putative role in the current imaging geometry and the chosen reconstruction technique, we developed an attenuation compensated Born-normalization method to reduce the attenuation effects and cancel off experimental factors when collecting quantitative fluorescence datasets over large area. Results of both simulation and phantom study demonstrated that fluorescent targets could be recovered accurately and quantitatively using this reconstruction mechanism. Finally, in vivo experiment confirms that the imaging system associated with the proposed image reconstruction approach was able to extract both functional and anatomical information, thereby improving quantification and localization of molecular targets.« less

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice.

PubMed

Morino, Kazuko; Kimizu, Mayumi; Fujiwara, Masayuki

2016-01-01

Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys 140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

PubMed

Wiley, L M; Obasaju, M F; Overstreet, J W; Cross, N L; Hanson, F W; Chang, R J

1987-08-01

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Sun-induced fluorescence - a new probe of photosynthesis: First maps from the imaging spectrometer HyPlant.

PubMed

Rascher, U; Alonso, L; Burkart, A; Cilia, C; Cogliati, S; Colombo, R; Damm, A; Drusch, M; Guanter, L; Hanus, J; Hyvärinen, T; Julitta, T; Jussila, J; Kataja, K; Kokkalis, P; Kraft, S; Kraska, T; Matveeva, M; Moreno, J; Muller, O; Panigada, C; Pikl, M; Pinto, F; Prey, L; Pude, R; Rossini, M; Schickling, A; Schurr, U; Schüttemeyer, D; Verrelst, J; Zemek, F

2015-12-01

Variations in photosynthesis still cause substantial uncertainties in predicting photosynthetic CO2 uptake rates and monitoring plant stress. Changes in actual photosynthesis that are not related to greenness of vegetation are difficult to measure by reflectance based optical remote sensing techniques. Several activities are underway to evaluate the sun-induced fluorescence signal on the ground and on a coarse spatial scale using space-borne imaging spectrometers. Intermediate-scale observations using airborne-based imaging spectroscopy, which are critical to bridge the existing gap between small-scale field studies and global observations, are still insufficient. Here we present the first validated maps of sun-induced fluorescence in that critical, intermediate spatial resolution, employing the novel airborne imaging spectrometer HyPlant. HyPlant has an unprecedented spectral resolution, which allows for the first time quantifying sun-induced fluorescence fluxes in physical units according to the Fraunhofer Line Depth Principle that exploits solar and atmospheric absorption bands. Maps of sun-induced fluorescence show a large spatial variability between different vegetation types, which complement classical remote sensing approaches. Different crop types largely differ in emitting fluorescence that additionally changes within the seasonal cycle and thus may be related to the seasonal activation and deactivation of the photosynthetic machinery. We argue that sun-induced fluorescence emission is related to two processes: (i) the total absorbed radiation by photosynthetically active chlorophyll; and (ii) the functional status of actual photosynthesis and vegetation stress. © 2015 John Wiley & Sons Ltd.

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells.

PubMed

Wüstner, Daniel; Lund, Frederik W; Röhrl, Clemens; Stangl, Herbert

2016-01-01

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy

PubMed Central

Arkhipov, Anton; Hüve, Jana; Kahms, Martin; Peters, Reiner; Schulten, Klaus

2007-01-01

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved. PMID:17704168

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

PubMed Central

Shcherbakova, Daria M.; Baloban, Mikhail; Emelyanov, Alexander V.; Brenowitz, Michael; Guo, Peng; Verkhusha, Vladislav V.

2016-01-01

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes. PMID:27539380

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source

PubMed Central

Pompidor, Guillaume; Dworkowski, Florian S. N.; Thominet, Vincent; Schulze-Briese, Clemens; Fuchs, Martin R.

2013-01-01

The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years. PMID:23955041

A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source.

PubMed

Pompidor, Guillaume; Dworkowski, Florian S N; Thominet, Vincent; Schulze-Briese, Clemens; Fuchs, Martin R

2013-09-01

The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years.

Pre-transplant donor HLA-specific antibodies: characteristics causing detrimental effects on survival after lung transplantation.

PubMed

Smith, John D; Ibrahim, Mohamed W; Newell, Helen; Danskine, Anna J; Soresi, Simona; Burke, Margaret M; Rose, Marlene L; Carby, Martin

2014-10-01

The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non-donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients (p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non-complement-fixing DSA, 75.8% for non-complement-fixing NDSA and 71.8% for antibody-negative patients (p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 (p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year (p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA (p < 0.0001, HR = 11.083) and DSA with MFI >5,000 (p = 0.0001, HR = 5.512). Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients. Copyright © 2014 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.

PubMed

Bellone, S; Roque, D; Cocco, E; Gasparrini, S; Bortolomai, I; Buza, N; Abu-Khalaf, M; Silasi, D-A; Ratner, E; Azodi, M; Schwartz, P E; Rutherford, T J; Pecorelli, S; Santin, A D

2012-04-24

We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro. Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays. High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu. Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy.

PubMed

Thomé, Marcos P; Filippi-Chiela, Eduardo C; Villodre, Emilly S; Migliavaca, Celina B; Onzi, Giovana R; Felipe, Karina B; Lenz, Guido

2016-12-15

Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods. © 2016. Published by The Company of Biologists Ltd.

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

PubMed

Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

2017-05-26

Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Amplified emission and modified spectral features in an opal hetero-structure mediated by passive defect mode localization

NASA Astrophysics Data System (ADS)

Rout, Dipak; Kumar, Govind; Vijaya, R.

2018-01-01

A photonic crystal hetero-structure consisting of a passive planar defect of SiO2 thin film sandwiched between two identical opals grown by inward growing self-assembly method using Rhodamine-B dye-doped polystyrene microspheres is studied for the characteristics of dye emission. The optical properties and the defect mode characteristics of the hetero-structure are studied from the reflection and transmission measurements. Laser-induced fluorescence from the hetero-structure showed amplified and spectrally narrowed emission compared to the photonic crystal emphasizing the role of the defect mode and distributed feedback. The enhanced emission is also complemented by the reduction in fluorescence decay time in the case of the hetero-structure in comparison to the 3D photonic crystals.

Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport.

PubMed

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-02-17

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level.

Particle Image Velocimetry Applications Using Fluorescent Dye-Doped Particles

NASA Technical Reports Server (NTRS)

Petrosky, Brian J.; Maisto, Pietro; Lowe, K. Todd; Andre, Matthieu A.; Bardet, Philippe M.; Tiemsin, Patsy I.; Wohl, Christopher J.; Danehy, Paul M.

2015-01-01

Polystyrene latex sphere particles are widely used to seed flows for velocimetry techniques such as Particle Image Velocimetry (PIV) and Laser Doppler Velocimetry (LDV). These particles may be doped with fluorescent dyes such that signals spectrally shifted from the incident laser wavelength may be detected via Laser Induced Fluorescence (LIF). An attractive application of the LIF signal is achieving velocimetry in the presence of strong interference from laser scatter, opening up new research possibilities very near solid surfaces or at liquid/gas interfaces. Additionally, LIF signals can be used to tag different fluid streams to study mixing. While fluorescence-based PIV has been performed by many researchers for particles dispersed in water flows, the current work is among the first in applying the technique to micron-scale particles dispersed in a gas. A key requirement for such an application is addressing potential health hazards from fluorescent dyes; successful doping of Kiton Red 620 (KR620) has enabled the use of this relatively safe dye for fluorescence PIV for the first time. In this paper, basic applications proving the concept of PIV using the LIF signal from KR620-doped particles are exhibited for a free jet and a twophase flow apparatus. Results indicate that while the fluorescence PIV techniques are roughly 2 orders of magnitude weaker than Mie scattering, they provide a viable method for obtaining data in flow regions previously inaccessible via standard PIV. These techniques have the potential to also complement Mie scattering signals, for example in multi-stream and/or multi-phase experiments.

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al, Hui-wang; Henderson, J. Nathan; Remington, S. James

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identifiedmore » dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 {angstrom} crystal structure (1 {angstrom}=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.« less

Quantification of Coffea arabica and Coffea canephora var. robusta concentration in blends by means of synchronous fluorescence and UV-Vis spectroscopies.

PubMed

Dankowska, A; Domagała, A; Kowalewski, W

2017-09-01

The potential of fluorescence, UV-Vis spectroscopies as well as the low- and mid-level data fusion of both spectroscopies for the quantification of concentrations of roasted Coffea arabica and Coffea canephora var. robusta in coffee blends was investigated. Principal component analysis was used to reduce data multidimensionality. To calculate the level of undeclared addition, multiple linear regression (PCA-MLR) models were used with lowest root mean square error of calibration (RMSEC) of 3.6% and root mean square error of cross-validation (RMSECV) of 7.9%. LDA analysis was applied to fluorescence intensities and UV spectra of Coffea arabica, canephora samples, and their mixtures in order to examine classification ability. The best performance of PCA-LDA analysis was observed for data fusion of UV and fluorescence intensity measurements at wavelength interval of 60nm. LDA showed that data fusion can achieve over 96% of correct classifications (sensitivity) in the test set and 100% of correct classifications in the training set, with low-level data fusion. The corresponding results for individual spectroscopies ranged from 90% (UV-Vis spectroscopy) to 77% (synchronous fluorescence) in the test set, and from 93% to 97% in the training set. The results demonstrate that fluorescence, UV, and visible spectroscopies complement each other, giving a complementary effect for the quantification of roasted Coffea arabica and Coffea canephora var. robusta concentration in blends. Copyright © 2017 Elsevier B.V. All rights reserved.

X-ray fluorescence measurements of dissolved gas and cavitation

DOE PAGES

Duke, Daniel J.; Kastengren, Alan L.; Swantek, Andrew B.; ...

2016-09-28

The dynamics of dissolved gas and cavitation are strongly coupled, yet these phenomena are difficult to measure in-situ. Both create voids in the fluid that can be difficult to distinguish. In this paper, we present an application of X-ray fluorescence in which liquid density and total noncondensible gas concentration (both dissolved and nucleated) are simultaneously measured. The liquid phase is doped with 400 ppm of a bromine tracer, and dissolved air is removed and substituted with krypton. Fluorescent emission at X-ray wavelengths is simultaneously excited from the Br and Kr with a focused monochromatic X-ray beam from a synchrotron source.more » We measure the flow in a cavitating nozzle 0.5 mm in diameter. From Br fluorescence, total displacement of the liquid is measured. From Kr fluorescence, the mass fraction of both dissolved and nucleated gas is measured. Volumetric displacement of liquid due to both cavitation and gas precipitation can be separated through estimation of the local equilibrium dissolved mass fraction. The uncertainty in the line of sight projected densities of the liquid and gas phases is 4–6 %. The high fluorescence yields and energies of Br and Kr allow small mass fractions of gas to be measured, down to 10 -5, with an uncertainty of 8 %. Finally, these quantitative measurements complement existing optical diagnostic techniques and provide new insight into the diffusion of gas into cavitation bubbles, which can increase their internal density, pressure and lifetimes by orders of magnitude.« less

Optimisation of fluorescence guidance during robot-assisted laparoscopic sentinel node biopsy for prostate cancer.

PubMed

KleinJan, Gijs H; van den Berg, Nynke S; Brouwer, Oscar R; de Jong, Jeroen; Acar, Cenk; Wit, Esther M; Vegt, Erik; van der Noort, Vincent; Valdés Olmos, Renato A; van Leeuwen, Fijs W B; van der Poel, Henk G

2014-12-01

The hybrid tracer was introduced to complement intraoperative radiotracing towards the sentinel nodes (SNs) with fluorescence guidance. Improve in vivo fluorescence-based SN identification for prostate cancer by optimising hybrid tracer preparation, injection technique, and fluorescence imaging hardware. Forty patients with a Briganti nomogram-based risk >10% of lymph node (LN) metastases were included. After intraprostatic tracer injection, SN mapping was performed (lymphoscintigraphy and single-photon emission computed tomography with computed tomography (SPECT-CT)). In groups 1 and 2, SNs were pursued intraoperatively using a laparoscopic gamma probe followed by fluorescence imaging (FI). In group 3, SNs were initially located via FI. Compared with group 1, in groups 2 and 3, a new tracer formulation was introduced that had a reduced total injected volume (2.0 ml vs. 3.2 ml) but increased particle concentration. For groups 1 and 2, the Tricam SLII with D-Light C laparoscopic FI (LFI) system was used. In group 3, the LFI system was upgraded to an Image 1 HUB HD with D-Light P system. Hybrid tracer-based SN biopsy, extended pelvic lymph node dissection, and robot-assisted radical prostatectomy. Number and location of the preoperatively identified SNs, in vivo fluorescence-based SN identification rate, tumour status of SNs and LNs, postoperative complications, and biochemical recurrence (BCR). Mean fluorescence-based SN identification improved from 63.7% (group 1) to 85.2% and 93.5% for groups 2 and 3, respectively (p=0.012). No differences in postoperative complications were found. BCR occurred in three pN0 patients. Stepwise optimisation of the hybrid tracer formulation and the LFI system led to a significant improvement in fluorescence-assisted SN identification. Preoperative SPECT-CT remained essential for guiding intraoperative SN localisation. Intraoperative fluorescence-based SN visualisation can be improved by enhancing the hybrid tracer formulation and laparoscopic fluorescence imaging system. Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

PubMed Central

2015-01-01

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein–protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein–protein interactions within whole animals. PMID:25265085

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

PubMed

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Mathematical and computational approaches can complement experimental studies of host-pathogen interactions.

PubMed

Kirschner, Denise E; Linderman, Jennifer J

2009-04-01

In addition to traditional and novel experimental approaches to study host-pathogen interactions, mathematical and computer modelling have recently been applied to address open questions in this area. These modelling tools not only offer an additional avenue for exploring disease dynamics at multiple biological scales, but also complement and extend knowledge gained via experimental tools. In this review, we outline four examples where modelling has complemented current experimental techniques in a way that can or has already pushed our knowledge of host-pathogen dynamics forward. Two of the modelling approaches presented go hand in hand with articles in this issue exploring fluorescence resonance energy transfer and two-photon intravital microscopy. Two others explore virtual or 'in silico' deletion and depletion as well as a new method to understand and guide studies in genetic epidemiology. In each of these examples, the complementary nature of modelling and experiment is discussed. We further note that multi-scale modelling may allow us to integrate information across length (molecular, cellular, tissue, organism, population) and time (e.g. seconds to lifetimes). In sum, when combined, these compatible approaches offer new opportunities for understanding host-pathogen interactions.

A new karyotype for Rhipidomys (Rodentia, Cricetidae) from Southeastern Brazil

PubMed Central

de Carvalho, Ana Heloisa; Lopes, Maria Olímpia Garcia; Svartman, Marta

2012-01-01

Abstract In this work we present a new karyotype for Rhipidomys Tschudi, 1845 (Cricetidae, Rodentia) from Brazil. Our chromosome analyses included GTG- and CBG-banding patterns, the localization of the nucleolus organizer regions after silver staining (Ag-NORs) and fluorescence in situ hybridization (FISH) with a telomere probe. The new karyotype is composed of 44 chromosomes and has a fundamental number (number of autosomal arms) of 48. Most Rhipidomys species already karyotyped presented similar complements with 2n=44, but their fundamental numbers varied from FN=46 to 80, a variation that has been mainly attributed to pericentric inversions. The comparison of this new karyotype to those of other Rhipidomys already reported allowed us to conclude that it is a distinctive chromosome complement, which can be of great use as a tool for the very complicated taxonomic identification in this genus. PMID:24260664

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with onemore » GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.« less

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

PubMed

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis, Photophysical, and Biological Evaluation of Sulfated Polyglycerol Dendronized Perylenebisimides (PBIs)--A Promising Platform for Anti-Inflammatory Theranostic Agents?

PubMed

Heek, T; Kühne, C; Depner, H; Achazi, K; Dernedde, J; Haag, R

2016-03-16

A set of four water-soluble perylene bisimides (PBI) based on sulfated polyglycerol (PGS) dendrons were developed, their photophysical properties determined via UV/vis and fluorescence spectroscopy, and their performance as possible anti-inflammatory agents evaluated via biological in vitro studies. It could be shown that in contrast to charge neutral PG-PBIs the introduction of the additional electrostatic repulsion forces leads to a decrease in the dendron generation necessary for aggregation suppression, allowing the preparation of PBIs with fluorescence quantum yields of >95% with a considerable decreased synthetic effort. Furthermore, the values determined for L-selectin binding down to the nanomolar range, their limited impact on blood coagulation, and their minor activation of the complement system renders these systems ideal for anti-inflammatory purposes.

Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport*

PubMed Central

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-01-01

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level. PMID:22190681

X-Ray Fluorescence to Determine Zn in Bolivian Children using Hair Samples

NASA Astrophysics Data System (ADS)

Tellería Narvaez, C. A.; Fernández Alcázar, S.; Barrientos Zamora, F. G.; Chungara Castro, J.; Luna Lauracia, I.; Mamani Tola, H.; Mita Peralta, E.; Muñoz Gosálvez, A. O.; Romero Bolaños, L. E.; Ramírez Ávila, G. M.

2014-06-01

As a first step in the evaluation of nutritional levels in Bolivian children (8-13 years-old), we carried out X-Ray Fluorescence measurements in hair samples of children belonging to different social classes and living either in rural areas or in cities. The aim of this study is to contribute to health policies tending to improve the global health of children and consequently avoid malnutrition. Our method intends to have maximum reliability and at the same time be as simple as possible from an experimental point of view. Additionally, we use this method to determine some other elements such as Fe, Cu, Pb, As and Hg, the latter three considered as contaminants that could be present in children living in areas which neighbor mines and industries. This work will be complemented by some biological and medical tests.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

New insights into heat induced structural changes of pectin methylesterase on fluorescence spectroscopy and molecular modeling basis

NASA Astrophysics Data System (ADS)

Nistor, Oana Viorela; Stănciuc, Nicoleta; Aprodu, Iuliana; Botez, Elisabeta

2014-07-01

Heat-induced structural changes of Aspergillus oryzae pectin methylesterase (PME) were studied by means of fluorescence spectroscopy and molecular modeling, whereas the functional enzyme stability was monitored by inactivation studies. The fluorescence spectroscopy experiments were performed at two pH value (4.5 and 7.0). At both pH values, the phase diagrams were linear, indicating the presence of two molecular species induced by thermal treatment. A red shift of 7 nm was observed at neutral pH by increasing temperature up to 60 °C, followed by a blue shift of 4 nm at 70 °C, suggesting significant conformational rearrangements. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of enzyme with increasing temperature, especially at neutral pH. The experimental results were complemented with atomic level observations on PME model behavior after performing molecular dynamics simulations at different temperatures. The inactivation kinetics of PME in buffer solutions was fitted using a first-order kinetics model, resulting in activation energy of 241.4 ± 7.51 kJ mol-1.

CARS microscopy of Alzheimer's diseased brain tissue

NASA Astrophysics Data System (ADS)

Enejder, Annika; Kiskis, Juris; Fink, Helen; Nyberg, Lena; Thyr, Jakob; Li, Jia-Yi

2014-02-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

A validation study of the 1,2-indandione reagent for operational use in the UK: Part 2 - Optimization of processing conditions.

PubMed

Nicolasora, Niko; Downham, Rory; Dyer, Rachel-May; Hussey, Laura; Luscombe, Aoife; Sears, Vaughn

2018-05-02

This paper contains details of work carried out to identify the most effective processing conditions for the optimized 1,2-indandione/zn formulation developed for use under UK conditions. Using direct measurements of fluorescence taken from test spots of amino acids and eccrine sweat during oven processing, complemented with experiments on real fingermarks, it was established that processing temperatures above 120°C in the oven were detrimental to the fluorescence of the developed mark. Alternative methods of development to oven processing were found to be effective, but less controllable. High levels of humidification were also found to be detrimental to the fluorescence of 1,2-indandione developed marks, and oven processing at 100°C and 0% relative humidity is therefore recommended for further studies. It has also been shown that 1,2-indandione can develop fingermarks at temperatures as low as 20°C, making it a candidate for use at crime scenes. Copyright © 2018. Published by Elsevier B.V.

Garlic virus X 11-kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus.

PubMed

Lu, Yuwen; Yan, Fei; Guo, Wei; Zheng, Hongying; Lin, Lin; Peng, Jiejun; Adams, Michael J; Chen, Jianping

2011-09-01

The subcellular localization of the 11-kDa protein (p11) encoded by ORF3 of Garlic virus X (GarVX; genus Allexivirus, family Alphaflexiviridae) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells of Nicotiana benthamiana. Moreover, the p11-RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17-kDa protein (p17) of garlic interacting with p11 was identified by yeast two-hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells of N. benthamiana, it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD. NO CLAIM TO ORIGINAL US GOVERNMENT WORKS.

Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2016-11-15

Interaction of the food colorant tartrazine with human hemoglobin was studied using multispectroscopic and microcalorimetric techniques to gain insights into the binding mechanism and thereby the toxicity aspects. Hemoglobin spectrum showed hypochromic changes in the presence of tartrazine. Quenching of the fluorescence of hemoglobin occurred and the quenching mechanism was through a static mode as revealed from temperature dependent and time-resolved fluorescence studies. According to the FRET theory the distance between β-Trp37 of hemoglobin and bound tartrazine was evaluated to be 3.44nm. Synchronous fluorescence studies showed that tartrazine binding led to alteration of the microenvironment around the tryptophans more in comparison to tyrosines. 3D fluorescence and FTIR data provided evidence for conformational changes in the protein on binding. Circular dichroism studies revealed that the binding led to significant loss in the helicity of hemoglobin. The esterase activity assay further complemented the circular dichroism data. Microcalorimetric study using isothermal titration calorimetry revealed the binding to be exothermic and driven largely by positive entropic contribution. Dissection of the Gibbs energy change proposed the protein-dye complexation to be dominated by non-polyelectrolytic forces. Negative heat capacity change also corroborated the involvement of hydrophobic forces in the binding process. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Toward Fourier interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Kohen, Elli; Hirschberg, Joseph G.; Berry, John P.; Ozkutuk, Nuri; Ornek, Ceren; Monti, Marco; Leblanc, Roger M.; Schachtschabel, Dietrich O.; Haroon, Sumaira

2003-10-01

Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and other osteosarcoma, human breast and color cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR,USA; a recently synthesized mitochondrial oxygen probe, [PRE,P1"- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in teh blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochonrial distribution and morphology. In terms of application to biomedicine (for diagnostiscs, prognostsics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnical applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.

In vivo X-ray fluorescence of lead in bone: review and current issues.

PubMed Central

Todd, A C; Chettle, D R

1994-01-01

Bone lead measurements can assess long-term lead dosimetry because the residence time of lead in bone is long. Bone lead measurements thus complement blood and plasma lead measurements, which reflect more short-term exposure. Although the noninvasive, in vivo measurement of lead in bone by X-ray fluorescence (XRF) has been under development since the 1970s, its use is still largely confined to research institutions. There are three principal methods used that vary both in the how lead X-rays are fluoresced and in which lead X-rays are fluoresced. Several groups have reported the independent development of in vivo measurement systems, the majority adopting the 109Cd K XRF method because of its advantages: a robust measurement, a lower detection limit (compared to 57Co K XRF), and a lower effective (radiation) dose (compared to L XRF) when calculated according to the most recent guidelines. These advantages, and the subsequent widespread adoption of the 109Cd method, are primarily consequences of the physics principles of the technique. This paper presents an explanation of the principles of XRF, a description of the practical measurement systems, a review of the human bone lead studies performed to date; and a discussion of some issues surrounding future application of the methods. Images p172-a PMID:8033846

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duke, Daniel J.; Kastengren, Alan L.; Swantek, Andrew B.

The dynamics of dissolved gas and cavitation are strongly coupled, yet these phenomena are difficult to measure in-situ. Both create voids in the fluid that can be difficult to distinguish. In this paper, we present an application of X-ray fluorescence in which liquid density and total noncondensible gas concentration (both dissolved and nucleated) are simultaneously measured. The liquid phase is doped with 400 ppm of a bromine tracer, and dissolved air is removed and substituted with krypton. Fluorescent emission at X-ray wavelengths is simultaneously excited from the Br and Kr with a focused monochromatic X-ray beam from a synchrotron source.more » We measure the flow in a cavitating nozzle 0.5 mm in diameter. From Br fluorescence, total displacement of the liquid is measured. From Kr fluorescence, the mass fraction of both dissolved and nucleated gas is measured. Volumetric displacement of liquid due to both cavitation and gas precipitation can be separated through estimation of the local equilibrium dissolved mass fraction. The uncertainty in the line of sight projected densities of the liquid and gas phases is 4–6 %. The high fluorescence yields and energies of Br and Kr allow small mass fractions of gas to be measured, down to 10 -5, with an uncertainty of 8 %. Finally, these quantitative measurements complement existing optical diagnostic techniques and provide new insight into the diffusion of gas into cavitation bubbles, which can increase their internal density, pressure and lifetimes by orders of magnitude.« less

A comparison of the absorbed fluorescent treponemal antibody (FTA-ABS) test and other screening tests for treponemal disease in patients attending a venereal disease clinic.

PubMed

Wilkinson, A E; Scrimgeour, G; Rodin, P

1972-05-01

Screening tests-absorbed fluorescent treponemal (FTA-ABS), the Reiter protein complement-fixation (RPCFT), VDRL slide test, automated reagin-and cardiolipin Wassermann reaction-were carried out on 1922 consecutive new patients attending the Whitechapel Clinic over a three-month period.Taking the FTA-ABS test results as an index, the most efficient combination of conventional tests was found to be the RPCFT and automated reagin test. The cardiolipin WR proved to be under-sensitive and of little value compared with the other tests.Forty-two per cent of the 107 sera reactive in the FTA-ABS test were not detected by the RPCFT or ART tests. An assessment based on the TPI test results and clinical findings in these patients is presented. The scope and limitations of the FTA-ABS test as a screening procedure are discussed.

Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space

NASA Astrophysics Data System (ADS)

Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

2014-07-01

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

Super-resolution imaging and tracking of protein–protein interactions in sub-diffraction cellular space

PubMed Central

Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

2014-01-01

Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein–protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB–EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB–EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB–EF-Tu interactions. PMID:25030837

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

PubMed Central

Nilsson, Mats; Gullberg, Mats; Dahl, Fredrik; Szuhai, Karoly; Raap, Anton K.

2002-01-01

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color. PMID:12136114

Chlamydia-induced septic polyarthritis in a dog.

PubMed

Lambrechts, N; Picard, J; Tustin, R C

1999-03-01

A systemic disease associated with pyrexia, lymphadenopathy, and arthropathy of several joints of the appendicular skeleton in a dog is described. Chlamydia-like organisms were detected on light-microscopic examination of a smear made from joint fluid aspirated from one of the affected joints. A group-specific lipopolysaccharide antigen shared by all Chlamydia spp. was demonstrated by direct fluorescent antibody staining of joint fluid, which also proved positive for chlamydia by means of the relevant polymerase chain reaction test. An indirect fluorescent antibody test on serum was also positive, although the complement fixation test was negative. Attempts to grow the organism from joint aspirates in the yolk sac of embryonating hens' eggs and on appropriate tissue cultures, however, failed. Chlamydia spp. are considered to have played an aetiological role in this case, making it the first substantiated case of naturally-occurring arthropathy in a dog due to chlamydiosis. The origin of the infection could not be traced.

Co-design in synthetic biology: a system-level analysis of the development of an environmental sensing device.

PubMed

Ball, David A; Lux, Matthew W; Graef, Russell R; Peterson, Matthew W; Valenti, Jane D; Dileo, John; Peccoud, Jean

2010-01-01

The concept of co-design is common in engineering, where it is necessary, for example, to determine the optimal partitioning between hardware and software of the implementation of a system features. Here we propose to adapt co-design methodologies for synthetic biology. As a test case, we have designed an environmental sensing device that detects the presence of three chemicals, and returns an output only if at least two of the three chemicals are present. We show that the logical operations can be implemented in three different design domains: (1) the transcriptional domain using synthetically designed hybrid promoters, (2) the protein domain using bi-molecular fluorescence complementation, and (3) the fluorescence domain using spectral unmixing and relying on electronic processing. We discuss how these heterogeneous design strategies could be formalized to develop co-design algorithms capable of identifying optimal designs meeting user specifications.

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

PubMed Central

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

X-Ray Fluorescence to Determine Zn in Bolivian Children using Hair Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tellería Narvaez, C.A.; Fernández Alcázar, S.; Barrientos Zamora, F.G.

As a first step in the evaluation of nutritional levels in Bolivian children (8–13 years-old), we carried out X-Ray Fluorescence measurements in hair samples of children belonging to different social classes and living either in rural areas or in cities. The aim of this study is to contribute to health policies tending to improve the global health of children and consequently avoid malnutrition. Our method intends to have maximum reliability and at the same time be as simple as possible from an experimental point of view. Additionally, we use this method to determine some other elements such as Fe, Cu,more » Pb, As and Hg, the latter three considered as contaminants that could be present in children living in areas which neighbor mines and industries. This work will be complemented by some biological and medical tests.« less

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

DNA transformation via local heat shock

NASA Astrophysics Data System (ADS)

Li, Sha; Meadow Anderson, L.; Yang, Jui-Ming; Lin, Liwei; Yang, Haw

2007-07-01

This work describes transformation of foreign DNA into bacterial host cells by local heat shock using a microfluidic system with on-chip, built-in platinum heaters. Plasmid DNA encoding ampicillin resistance and a fluorescent protein can be effectively transformed into the DH5α chemically competent E. coli using this device. Results further demonstrate that only one-thousandth of volume is required to obtain transformation efficiencies as good as or better than conventional practices. As such, this work complements other lab-on-a-chip technologies for potential gene cloning/therapy and protein expression applications.

Mirror image DNA nanostructures for chiral supramolecular assemblies.

PubMed

Lin, Chenxiang; Ke, Yonggang; Li, Zhe; Wang, James H; Liu, Yan; Yan, Hao

2009-01-01

L-DNA, the mirror image of natural D-DNA, can be readily self-assembled into designer discrete or periodic nanostructures. The assembly products are characterized by polyacrylamide gel electrophoresis, circular dichroism spectrum, atomic force microscope, and fluorescence microscope. We found that the use of enantiomer DNA as building material leads to the formation of DNA supramolecules with opposite chirality. Therefore, the L-DNA self-assembly is a substantial complement to the structural DNA nanotechnology. Moreover, the L-DNA architectures feature superior nuclease resistance thus are appealing for in vivo medical applications.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

PubMed Central

Johnson, M. Brittany; Criss, Alison K.

2013-01-01

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

PubMed Central

Marriott, G.; Heidecker, M.; Diamandis, E. P.; Yan-Marriott, Y.

1994-01-01

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found. Through the coupling of SBMC to streptavidin,a plethora of biotin-based tracer molecules are available for immunocytochemical studies. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7811952

Sounding rocket flight report: MUMP 9 and MUMP 10

NASA Technical Reports Server (NTRS)

Grassl, H. J.

1971-01-01

The results of the launching of two Marshall-University of Michigan Probes (MUMP 9 and MUMP 10), Nike-Tomahawk sounding rocket payloads, are summarized. The MUMP 9 paylaod included an omegatron mass analyzer, a molecular fluorescence densitometer, a mini-tilty filter, and a lunar position sensor. This complement of instruments permitted the determination of the molecular nitrogen density and temperature in the altitude range from approximately 143 to 297 km over Wallops Island, Virginia, during January 1971. The MUMP 10 payload included an omegatron mass analyzer, an electron temperature probe (Spencer, Brace, and Carignan, 1962), a cryogenic densitometer, and a solar position sensor. This complement of instruments permitted the determination of the molecular nitrogen density and temperature and the charged particle density and temperature in the altitude range from approximately 145 to 290 km over Wallops Island, Virginia, during the afternoon preceding the MUMP 9 launch in January 1971. A general description of the payload kinematics, orientation analysis, and the technique for the reduction and analysis of the data is given.

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

PubMed

Masihzadeh, Omid; Ammar, David A; Kahook, Malik Y; Lei, Tim C

2013-05-01

To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

Synthetic fluorescent probes for studying copper in biological systems

PubMed Central

Cotruvo, Joseph A.; Aron, Allegra T.; Ramos-Torres, Karla M.; Chang, Christopher J.

2015-01-01

The potent redox activity of copper is required for sustaining life. Mismanagement of its cellular pools, however, can result in oxidative stress and damage connected to aging, neurodegenerative diseases, and metabolic disorders. Therefore, copper homeostasis is tightly regulated by cells and tissues. Whereas copper and other transition metal ions are commonly thought of as static cofactors buried within protein active sites, emerging data points to the presence of additional loosely bound, labile pools that can participate in dynamic signalling pathways. Against this backdrop, we review advances in sensing labile copper pools and understanding their functions using synthetic fluorescent indicators. Following brief introductions to cellular copper homeostasis and considerations in sensor design, we survey available fluorescent copper probes and evaluate their properties in the context of their utility as effective biological screening tools. We emphasize the need for combined chemical and biological evaluation of these reagents, as well as the value of complementing probe data with other techniques for characterizing the different pools of metal ions in biological systems. This holistic approach will maximize the exciting opportunities for these and related chemical technologies in the study and discovery of novel biology of metals. PMID:25692243

Synthetic fluorescent probes for studying copper in biological systems.

PubMed

Cotruvo, Joseph A; Aron, Allegra T; Ramos-Torres, Karla M; Chang, Christopher J

2015-07-07

The potent redox activity of copper is required for sustaining life. Mismanagement of its cellular pools, however, can result in oxidative stress and damage connected to aging, neurodegenerative diseases, and metabolic disorders. Therefore, copper homeostasis is tightly regulated by cells and tissues. Whereas copper and other transition metal ions are commonly thought of as static cofactors buried within protein active sites, emerging data points to the presence of additional loosely bound, labile pools that can participate in dynamic signalling pathways. Against this backdrop, we review advances in sensing labile copper pools and understanding their functions using synthetic fluorescent indicators. Following brief introductions to cellular copper homeostasis and considerations in sensor design, we survey available fluorescent copper probes and evaluate their properties in the context of their utility as effective biological screening tools. We emphasize the need for combined chemical and biological evaluation of these reagents, as well as the value of complementing probe data with other techniques for characterizing the different pools of metal ions in biological systems. This holistic approach will maximize the exciting opportunities for these and related chemical technologies in the study and discovery of novel biology of metals.

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.

PubMed

Neville, David C A; Coquard, Virginie; Priestman, David A; te Vruchte, Danielle J M; Sillence, Daniel J; Dwek, Raymond A; Platt, Frances M; Butters, Terry D

2004-08-15

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

Tracking Seasonal and Diurnal Photosynthesis and Plant Water Status in Maize Using SIF, Eddy Covariance Fluxes, PAM Fluorescence and Gas Exchange

NASA Astrophysics Data System (ADS)

Chang, C.; Melkonian, J.; Riha, S. J.; Gu, L.; Sun, Y.

2017-12-01

Improving the sensitivity of methods for crop monitoring and yield forecasting is crucial as the frequency of extreme weather events increases. Conventional remote monitoring methods rely on greenness-based indices such as NDVI and EVI, which do not directly measure photosynthesis and are not sufficiently sensitive to rapid plant stress response. Solar-induced chlorophyll fluorescence (SIF) is a promising new technology that serves as a direct functional proxy of photosynthesis. We developed the first system utilizing dual QE Pro spectrometers to continuously measure the diurnal and seasonal cycle of SIF, and deployed the system in a corn field in upstate New York in 2017. To complement SIF, canopy-level measurements of carbon and water fluxes were also measured, along with concurrent leaf-level measurements of gas exchange and PAM fluorescence, midday water potential, leaf pigments, phenology, LAI, and soil moisture. We show that SIF is well correlated to GPP during the growing season and show that both are controlled by similar environmental conditions including PAR and water availability. We also describe diurnal changes in photosynthesis and plant water status and demonstrate the sensitivity of SIF to diurnal plant response.

Imaging molecular dynamics in vivo--from cell biology to animal models.

PubMed

Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I

2011-09-01

Advances in fluorescence microscopy have enabled the study of membrane diffusion, cell adhesion and signal transduction at the molecular level in living cells grown in culture. By contrast, imaging in living organisms has primarily been restricted to the localization and dynamics of cells in tissues. Now, imaging of molecular dynamics is on the cusp of progressing from cell culture to living tissue. This transition has been driven by the understanding that the microenvironment critically determines many developmental and pathological processes. Here, we review recent progress in fluorescent protein imaging in vivo by drawing primarily on cancer-related studies in mice. We emphasize the need for techniques that can be easily combined with genetic models and complement fluorescent protein imaging by providing contextual information about the cellular environment. In this Commentary we will consider differences between in vitro and in vivo experimental design and argue for an approach to in vivo imaging that is built upon the use of intermediate systems, such as 3-D and explant culture models, which offer flexibility and control that is not always available in vivo. Collectively, these methods present a paradigm shift towards the molecular-level investigation of disease and therapy in animal models of disease.

Close Encounters - Probing Proximal Proteins in Live or Fixed Cells.

PubMed

Lönn, Peter; Landegren, Ulf

2017-07-01

The well-oiled machinery of the cellular proteome operates via variable expression, modifications, and interactions of proteins, relaying genomic and transcriptomic information to coordinate cellular functions. In recent years, a number of techniques have emerged that serve to identify sets of proteins acting in close proximity in the course of orchestrating cellular activities. These proximity-dependent assays, including BiFC, BioID, APEX, FRET, and isPLA, have opened up new avenues to examine protein interactions in live or fixed cells. We review herein the current status of proximity-dependent in situ techniques. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research, and we discuss their potential as tools for drug development and diagnostics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Diversity and Evolution of Coral Fluorescent Proteins

PubMed Central

Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Beltran-Ramirez, Victor; Miller, David J.; Wiedenmann, Jörg; Salih, Anya; Matz, Mikhail V.

2008-01-01

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors. PMID:18648549

Chromosomal abnormalities in human sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, R.H.

1985-01-01

The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhapsmore » reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.« less

Post-mortem diagnosis of chronic Chagas's disease comparative evaluation of three serological tests on pericardial fluid.

PubMed

Lopes, E R; Chapadeiro, E; Batista, S M; Cunha, J G; Rocha, A; Miziara, L; Ribeiro, J U; Patto, R J

1978-01-01

In an attempt to improve the post-mortem diagnosis of Chagas's disease the authors performed haemagglutination tests (HAT), fluorescent Trypanosoma cruzi antibody tests (FAT), and complement fixation tests (CFT) on the pericardial fluid obtained at autopsy of 50 individuals with Chagas's heart disease, and 93 patients in whom this disease was not thought to be present. The results demonstrate that all three tests are efficient for the post-mortem diagnosis of Chagas's disease but suggest that their combined use would detect more cases than would one isolated reaction only.

46,XY/47,XYY/48,XYYY karyotype in a 3-year-old boy ascertained because of radioulnar synostosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, C.; Robson, L.; Jackson, J.

Chromosome analysis was performed on a 3-year-old boy because of bilateral radioulnar synostosis and demonstrated a mosaic karyotype 46,XY/47,XYY/48,XYYY. He had minor facial anomalies and mild intellectual delay. He appears to be the youngest patient reported with this rare chromosome complement. His father, mother, and brother had normal chromosomes. Fluorescence in situ hybridization (FISH) was performed on the propositus and his father with the Y chromosome heterochromatic probe (pHY3.4) to add to the evaluation of mosaicism. 17 refs., 3 figs., 2 tabs.

Optical, Fluorescence with quantum analysis of hydrazine (1, 3- Dinitro Phenyl) by DFT and Ab initio approach

NASA Astrophysics Data System (ADS)

Cecily Mary Glory, D.; Sambathkumar, K.; Madivanane, R.; Velmurugan, G.; Gayathri, R.; Nithiyanantham, S.; Venkatachalapathy, M.; Rajkamal, N.

2018-07-01

Experimental and computational study of molecular structure, vibrational and UV-spectral analysis of Hydrazine (1, 3- Dinitrophenyl) (HDP) derivatives. The crystal was grown by slow cooling method and the crystalline perfection of single crystals was evaluated by high resolution X-ray diffractometry (HRXRD) using a multicrystal X-ray diffractometer. Fluorescence, FT-IR and FT-Raman spectra of HDP crystal were recorded. The assignments of the vibrational spectra have been carried out with the help of normal co-ordinate analysis (NCA) followed by scaled quantum force field methodology (SQMFF). NMR studies have confirmed respectively the crystal structure and functional groups of the grown crystal. The energy and oscillator strength calculated by Time-Dependent Density Functional Theory (TD-DFT) result complements the experimental findings. The calculated MESP, UV, HOMO-LUMO energies show that charge transfer done within the molecule. And various thermodynamic parameters are studied. Fukui determines the local reactive site of electrophilic, nucleophilic, descriptor.

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

Imaging transcription factors dynamics with advanced fluorescence microscopy methods.

PubMed

Verneri, Paula; Romero, Juan José; De Rossi, María Cecilia; Alvarez, Yanina; Oses, Camila; Guberman, Alejandra; Levi, Valeria

2018-05-10

Pluripotent stem cells (PSCs) are capable of self-renewing and producing all cell types derived from the three germ layers in response to developmental cues, constituting an important promise for regenerative medicine. Pluripotency depends on specific transcription factors (TFs) that induce genes required to preserve the undifferentiated state and repress other genes related to differentiation. The transcription machinery and regulatory components such as TFs are recruited dynamically on their target genes making it essential exploring their dynamics in living cells to understand the transcriptional output. Non-invasive and very sensitive fluorescence microscopy methods are making it possible visualizing the dynamics of TFs in living specimens, complementing the information extracted from studies in fixed specimens and bulk assays. In this work, we briefly describe the basis of these microscopy methods and review how they contributed to our knowledge of the function of TFs relevant to embryo development and cell differentiation in a variety of systems ranging from single cells to whole organisms. Copyright © 2017. Published by Elsevier B.V.

Using enzyme folding to explore the mechanism of therapeutic touch: a feasibility study.

PubMed

Strickland, Mallory L; Boylan, Helen M

2010-07-01

The goal of this research is to design a novel model using protein folding to study Therapeutic Touch, a noncontact form of energy manipulation healing. Presented is a feasibility study suggesting that the denaturation path of ribonuclease A may be a useful model to study the energy exchange underlying therapeutic touch. The folding of ribonuclease A serves as a controlled energy-requiring system in which energy manipulation can be measured by the degree of folding achieved. A kinetic assay and fluorescence spectroscopy are used to assess the enzyme-folding state. The data suggest that the kinetic assay is a useful means of assessing the degree of refolding, and specifically, the enzyme function. However, fluorescence spectroscopy was not shown to be an effective measurement of enzyme structure for the purposes of this work. More research is needed to assess the underlying mechanism of therapeutic touch to complement the existing studies. An enzyme-folding model may provide a useful means of studying the energy exchange in therapeutic touch.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Fetal damage after accidental polio vaccination of an immune mother

PubMed Central

Burton, A. E.; Robinson, E. T.; Harper, W. F.; Bell, E. J.; Boyd, J. F.

1984-01-01

Irreparable damage to the anterior horn cells of the cervical and thoracic cord was found in a 20-week-old fetus whose mother was immune to poliomyelitis before conceiving but who was inadvertently given oral polio vaccine at 18 weeks gestation. Polio neutralizing antibody titres in sera, taken before and after pregnancy, were identical and were at levels normally regarded as providing protection. Unsuccessful attempts were made to isolate poliovirus from extracts of fetal brain, lung, liver and placenta. Fluorescent antibody tests were performed on various levels of the central nervous system and on the left and right extensor forearm muscles. Specific positive fluorescence to poliovirus 2 and 3 antigens was detected at dorsal spinal cord level only. One positive result was seen with Coxsackie A9 antiserum and fresh guinea-pig complement in the inflammatory cells in the right extensor forearm muscles. This experience, as yet unexplained, underlines the importance of ensuring that women are not pregnant prior to oral polio vaccination. ImagesFigure 1.Figure 2. PMID:6747944

The coral immune response facilitates protection against microbes during tissue regeneration.

PubMed

van de Water, Jeroen A J M; Ainsworth, Tracy D; Leggat, William; Bourne, David G; Willis, Bette L; van Oppen, Madeleine J H

2015-07-01

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system-related genes involved in the Toll-like and NOD-like receptor signalling pathways and the lectin-complement system in three phases (<2, 4 and 10 days post-injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post-injury), as were levels of non-fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post-injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral-associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria-specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near-complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness. © 2015 John Wiley & Sons Ltd.

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy: A Novel Technique for Imaging the Retina

PubMed Central

Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Lei, Tim C.

2013-01-01

Purpose. To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Methods. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. Results. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. Conclusions. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice. PMID:23580484

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

PubMed

Görlitz, Frederik; Kelly, Douglas J; Warren, Sean C; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J; Stuhmeier, Frank; Neil, Mark A A; Tate, Edward W; Dunsby, Christopher; French, Paul M W

2017-01-18

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

Spectral response characterization of CdTe sensors of different pixel size with the IBEX ASIC

NASA Astrophysics Data System (ADS)

Zambon, P.; Radicci, V.; Trueb, P.; Disch, C.; Rissi, M.; Sakhelashvili, T.; Schneebeli, M.; Broennimann, C.

2018-06-01

We characterized the spectral response of CdTe sensors with different pixel sizes - namely 75, 150 and 300 μm - bonded to the latest generation IBEX single photon counting ASIC developed at DECTRIS, to detect monochromatic X-ray energy in the range 10-60 keV. We present a comparison of pulse height spectra recorded for several energies, showing the dependence on the pixel size of the non-trivial atomic fluorescence and charge sharing effects that affect the detector response. The extracted energy resolution, in terms of full width at half maximum or FWHM, ranges from 1.5 to 4 keV according to the pixel size and chip configuration. We devoted a careful analysis to the Quantum Efficiency and to the Spectral Efficiency - a newly-introduced measure that quantifies the impact of fluorescence and escape phenomena on the spectrum integrity in high- Z material based detectors. We then investigated the influence of the photon flux on the aforementioned quantities up to 180 ṡ 106 cts/s/mm2 and 50 ṡ 106 cts/s/mm2 for the 150 μm and 300 μm pixel case, respectively. Finally, we complemented the experimental data with analytical and with Monte Carlo simulations - taking into account the stochastic nature of atomic fluorescence - with an excellent agreement.

The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer.

PubMed

Zhuang, Zhihao; Yoder, Bonita L; Burgers, Peter M J; Benkovic, Stephen J

2006-02-21

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATPgammaS cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

PubMed Central

Warren, Sean C.; Alibhai, Dominic; West, Lucien; Kumar, Sunil; Alexandrov, Yuriy; Munro, Ian; Garcia, Edwin; McGinty, James; Talbot, Clifford; Serwa, Remigiusz A.; Thinon, Emmanuelle; da Paola, Vincenzo; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Tate, Edward W.; Dunsby, Christopher; French, Paul M. W.

2017-01-01

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set. PMID:28190060

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

PubMed

Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

PubMed

Mosqueira, V C; Legrand, P; Gulik, A; Bourdon, O; Gref, R; Labarre, D; Barratt, G

2001-11-01

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

[Pemphigus herpetiformis: a clinical variant of pemphigus vulgaris].

PubMed

Bauer, R; Orfanos, C E

1980-10-01

Two female patients are reported in whom the clinical picture closely corresponded to dermatitis herpetiformis Duhring or bullous pemphigoid, while acantholytic changes were present histologically. Indirect immunofluorescence revealed circulating pemphigus antibodies. Direct fluorescence showed deposits of IgG and complement in the intercellular space of the affected areas. One of the two patients responded well to DDS. There have been reports in the recent literature on bullous dermatoses, which can be regarded as morphological variants of pemphigus vulgaris. The term pemphigus herpetiformis is used here to designate these variants. The diagnosis can only be made by histological and immunological examinations in connection with the clinical picture.

Probing Chemical Properties of Interstitial Micro-fluids in Ice

NASA Astrophysics Data System (ADS)

Cheng, J.; Colussi, A. J.; Hoffmann, M. R.

2007-12-01

Liquid is present as microscopic channels in polycrystalline ice at sub-freezing and even sub-eutectic temperatures. Not only do chemicals tend to concentrate substantially in this microscopic liquid phase, but local physicochemical properties may also differ widely from the bulk counterparts, therefore critically affecting the thermodynamics and kinetics of chemical processes occurring in frozen media such as snow, frost, and frost- flowers. This phenomenon has important implications in atmospheric chemistry such as affecting the composition of the atmospheric boundary layer in snow-covered regions. A method using con-focal laser scanning microscope equipped with a cryostat has been developed to measure physicochemical properties of the microscopic liquid phase in ice that are not readily extrapolated from the bulk data. The experimental setup allows for monitoring the freezing process of an aqueous solution with a sub- second time resolution and a submicron 3D spatial resolution. The physicochemical properties (e.g. viscosity, polarity, and acidity) can, in theory, be deduced from features of the fluorescence spectra of particular fluorescent indicators. For example, the acidity change during the freezing and melting process of electrolyte solutions has been monitored in real time by a pH-dependent dual emission fluorescent probe C-SNARF-1. The effects of temperature, freezing rate, and added electrolytes such as ammonium sulfate, sodium chloride and zwitterions are also examined. The findings complement the theory and previous experimental evidence of freezing hydrolysis.

Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

PubMed Central

Sakai, Yasuyoshi; Koller, Antonius; Rangell, Linda K.; Keller, Gilbert A.; Subramani, Suresh

1998-01-01

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis. PMID:9566964

Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast.

PubMed

Hori, Akiko; Yoshida, Minoru; Ling, Feng

2011-05-01

Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Spectrophotometric determination of gold(III) in forensic and pharmaceutical samples and results complemented with ICP AES and EDXRF analysis

NASA Astrophysics Data System (ADS)

Nagaraja, Vani; Kumar, M. Kiran; Giddappa, Nagendrappa

2017-02-01

Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the system 1 it oxidizes o-dianisidine, in the system 2 it oxidizes catechol followed by its coupling with o-dianisidine, in the system 3 it oxidizes catechol followed by its coupling with aniline sulphate forming dye products with respective λmax 446 nm, 540 nm, and 505 nm. All the three systems were optimized and analytical parameters were calculated. The molar absorptivity values were 9.27 × 104, 1.97 × 104 and 1.62 × 104 respectively for the systems 1, 2 and 3 with the corresponding Sandell sensitivity values (μg cm- 2), 0.0021, 0.0096 and 0.011. The optimized systems were used for the determination of gold present in some forensic jewellery and pharmaceutical samples and the results obtained were compared with the results of all samples determined by Inductively Coupled Plasma - Atomic Emission Spectrometric method and a few of them were also complemented by Energy Dispersive X-Ray Fluorescent spectral analysis.

Spectrophotometric determination of gold(III) in forensic and pharmaceutical samples and results complemented with ICP AES and EDXRF analysis.

PubMed

Nagaraja, Vani; Kumar, M Kiran; Giddappa, Nagendrappa

2017-02-15

Spectrophotometric method with three systems were developed here for the determination of gold(III) using o-dianisidine, aniline sulphate and catechol. Gold(III),in the system 1 it oxidizes o-dianisidine, in the system 2 it oxidizes catechol followed by its coupling with o-dianisidine, in the system 3 it oxidizes catechol followed by its coupling with aniline sulphate forming dye products with respective λ max 446nm, 540nm, and 505nm. All the three systems were optimized and analytical parameters were calculated. The molar absorptivity values were 9.27×10 4 , 1.97×10 4 and 1.62×10 4 respectively for the systems 1, 2 and 3 with the corresponding Sandell sensitivity values (μgcm -2 ), 0.0021, 0.0096 and 0.011. The optimized systems were used for the determination of gold present in some forensic jewellery and pharmaceutical samples and the results obtained were compared with the results of all samples determined by Inductively Coupled Plasma - Atomic Emission Spectrometric method and a few of them were also complemented by Energy Dispersive X-Ray Fluorescent spectral analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunomodulatory activity and chemical characterisation of sangre de drago (dragon's blood) from Croton lechleri.

PubMed

Risco, Ester; Ghia, Felipe; Vila, Roser; Iglesias, José; Alvarez, Elida; Cañigueral, Salvador

2003-09-01

The immunomodulatory activity of the latex from Croton lechleri (sangre de drago) was determined by in vitro assays. Classical (CP) and alternative (AP) complement pathways activities were determined in human serum. Intracellular generation of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs) and monocytes, and phagocytosis of opsonised fluorescent microspheres were measured by flow cytometry. Free radical scavenging activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Activity on proliferation of murine lymphocytes was also investigated. In addition, anti-inflammatory activity was assayed in vivo by carrageenan-induced rat paw oedema test. Some of the activities were compared with those of the isolated alkaloid taspine. Sangre de drago from Croton lechleri showed immunomodulatory activity. It exhibited a potent inhibitory activity on CP and AP of complement system and inhibited the proliferation of activated T-cells. The latex showed free radical scavenging capacity. Depending on the concentration, it showed antioxidant or prooxidant properties, and stimulated or inhibited the phagocytosis. Moreover, the latex has strong anti-inflammatory activity when administered i. p. Taspine cannot be considered the main responsible for these activities, and other constituents, probably proanthocyanidins, should be also involved.

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development.

PubMed

Zhang, Wei; Zhou, Xin; Wen, Chi-Kuang

2012-06-01

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed.

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

PubMed Central

Zhang, Wei; Zhou, Xin; Wen, Chi-Kuang

2012-01-01

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed. PMID:22451723

Nanoscale materials for hyperthermal theranostics

NASA Astrophysics Data System (ADS)

Smith, Bennett E.; Roder, Paden B.; Zhou, Xuezhe; Pauzauskie, Peter J.

2015-04-01

Recently, the use of nanoscale materials has attracted considerable attention with the aim of designing personalized therapeutic approaches that can enhance both spatial and temporal control over drug release, permeability, and uptake. Potential benefits to patients include the reduction of overall drug dosages, enabling the parallel delivery of different pharmaceuticals, and the possibility of enabling additional functionalities such as hyperthermia or deep-tissue imaging (LIF, PET, etc.) that complement and extend the efficacy of traditional chemotherapy and surgery. This mini-review is focused on an emerging class of nanometer-scale materials that can be used both to heat malignant tissue to reduce angiogenesis and DNA-repair while simultaneously offering complementary imaging capabilities based on radioemission, optical fluorescence, magnetic resonance, and photoacoustic methods.

Assessment of MMP-2/-9 expression by fluorescence endoscopy for evaluation of anastomotic healing in a murine model of anastomotic leakage

PubMed Central

Twardy, Vanessa; Becker, Felix; Geyer, Christiane; Schwegmann, Katrin; Mohr, Annika; Faust, Andreas

2018-01-01

Background Disturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing. Methods Distal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE. Results With increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage. Conclusion During disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage. PMID:29566031

Assessment of MMP-2/-9 expression by fluorescence endoscopy for evaluation of anastomotic healing in a murine model of anastomotic leakage.

PubMed

Neumann, Philipp-Alexander; Twardy, Vanessa; Becker, Felix; Geyer, Christiane; Schwegmann, Katrin; Mohr, Annika; Faust, Andreas; Lenz, Philipp; Rijcken, Emile

2018-01-01

Disturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing. Distal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE. With increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage. During disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage.

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avonto, Cristina; Chittiboyina, Amar G.; Rua, Diego

2015-12-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles aftermore » incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow and critical parameters is presented. • The method could provide a useful tool to complement existing chemical assays.« less

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.

PubMed

Subach, Fedor V; Malashkevich, Vladimir N; Zencheck, Wendy D; Xiao, Hui; Filonov, Grigory S; Almo, Steven C; Verkhusha, Vladislav V

2009-12-15

Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photo-activatable GFP by providing a red super-resolution color. PAmCherry1 is originally "dark" but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C(alpha)-C(beta) bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C(alpha)-N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO(2) molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C(alpha)-C(beta) bond. The double bond extends the pi-conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.

Mapping microscopic order in plant and mammalian cells and tissues: novel differential polarization attachment for new generation confocal microscopes (DP-LSM)

NASA Astrophysics Data System (ADS)

Steinbach, G.; Pawlak, K.; Pomozi, I.; Tóth, E. A.; Molnár, A.; Matkó, J.; Garab, G.

2014-03-01

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples—e.g. cells and tissues—measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the ‘conventional’ imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.

Whole-brain spectroscopic MRI biomarkers identify infiltrating margins in glioblastoma patients

PubMed Central

Cordova, James S.; Shu, Hui-Kuo G.; Liang, Zhongxing; Gurbani, Saumya S.; Cooper, Lee A. D.; Holder, Chad A.; Olson, Jeffrey J.; Kairdolf, Brad; Schreibmann, Eduard; Neill, Stewart G.; Hadjipanayis, Constantinos G.; Shim, Hyunsuk

2016-01-01

Background The standard of care for glioblastoma (GBM) is maximal safe resection followed by radiation therapy with chemotherapy. Currently, contrast-enhanced MRI is used to define primary treatment volumes for surgery and radiation therapy. However, enhancement does not identify the tumor entirely, resulting in limited local control. Proton spectroscopic MRI (sMRI), a method reporting endogenous metabolism, may better define the tumor margin. Here, we develop a whole-brain sMRI pipeline and validate sMRI metrics with quantitative measures of tumor infiltration. Methods Whole-brain sMRI metabolite maps were coregistered with surgical planning MRI and imported into a neuronavigation system to guide tissue sampling in GBM patients receiving 5-aminolevulinic acid fluorescence-guided surgery. Samples were collected from regions with metabolic abnormalities in a biopsy-like fashion before bulk resection. Tissue fluorescence was measured ex vivo using a hand-held spectrometer. Tissue samples were immunostained for Sox2 and analyzed to quantify the density of staining cells using a novel digital pathology image analysis tool. Correlations among sMRI markers, Sox2 density, and ex vivo fluorescence were evaluated. Results Spectroscopic MRI biomarkers exhibit significant correlations with Sox2-positive cell density and ex vivo fluorescence. The choline to N-acetylaspartate ratio showed significant associations with each quantitative marker (Pearson's ρ = 0.82, P < .001 and ρ = 0.36, P < .0001, respectively). Clinically, sMRI metabolic abnormalities predated contrast enhancement at sites of tumor recurrence and exhibited an inverse relationship with progression-free survival. Conclusions As it identifies tumor infiltration and regions at high risk for recurrence, sMRI could complement conventional MRI to improve local control in GBM patients. PMID:26984746

Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerberick, G.F.; Ryan, C.A.; Von Bargen, E.C.

Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis responsemore » compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent.« less

Diketo modification of curcumin affects its interaction with human serum albumin.

PubMed

Shaikh, Shaukat Ali M; Singh, Beena G; Barik, Atanu; Ramani, Modukuri V; Balaji, Neduri V; Subbaraju, Gottumukkala V; Naik, Devidas B; Indira Priyadarsini, K

2018-06-15

Curcumin isoxazole (CI) and Curcumin pyrazole (CP), the diketo modified derivatives of Curcumin (CU) are metabolically more stable and are being explored for pharmacological properties. One of the requirements in such activities is their interaction with circulatory proteins like human serum albumin (HSA). To understand this, the interactions of CI and CP with HSA have been investigated employing absorption and fluorescence spectroscopy and the results are compared with that of CU. The respective binding constants of CP, CI and CU with HSA were estimated to be 9.3×10 5 , 8.4×10 5 and 2.5×10 5 M -1 , which decreased with increasing salt concentration in the medium. The extent of decrease in the binding constant was the highest in CP followed by CI and CU. This revealed that along with hydrophobic interaction other binding modes like electrostatic interactions operate between CP/CI/CU with HSA. Fluorescence quenching studies of HSA with these compounds suggested that both static and dynamic quenching mechanisms operate, where the contribution of static quenching is higher for CP and CI than that for CU. From fluorescence resonance energy transfer studies, the binding site of CU, CI and CP was found to be in domain IIA of HSA. CU was found to bind in closer proximity with Trp214 as compared to CI and CP and the same was responsible for efficient energy transfer and the same was also established by fluorescence anisotropy measurements. Furthermore docking simulation complemented the experimental observation, where both electrostatic as well as hydrophobic interactions were indicated between HSA and CP, CI and CU. This study is useful in designing more stable CU derivatives having suitable binding properties with proteins like HSA. Copyright © 2018 Elsevier B.V. All rights reserved.

Biophysical and computational comparison on the binding affinity of three important nutrients to β-lactoglobulin: folic acid, ascorbic acid and vitamin K3.

PubMed

Shahraki, Somaye; Heydari, Ali; Saeidifar, Maryam; Gomroki, Masoumeh

2017-11-06

Small globular protein, β-lactoglobulin (βLG), which has significant affinity toward many drugs, is the most abundant whey protein in milk. In this study, the interaction of βLG with three important nutrients, ascorbic acid (ASC), folic acid (FOL), and vitamin K3 (VK3) was investigated by spectroscopic methods (UV-visible and fluorescence) along with molecular docking technique. The results of fluorescence measurements showed that studied nutrients strongly quenched βLG fluorescence in static (FOL and ACS) or static-dynamic combined quenching (VK3) mode. The values of binding constants (K βLG-ASC  ~ 4.34 × 10 4  M -1 , K βLG-FOL ~ 1.67 × 10 4  M -1 and K βLG-VK3 ~ 13.49 × 10 4  M -1 at 310 K) suggested that VK3 and FOL had stronger binding affinity toward βLG than ASC. Thermodynamic analysis indicated that hydrophobic interactions are the major forces in the stability of FOL-βLG complex with enthalpy- and entropy-driving mode while, hydrogen bonds and van der Waals interactions play a major role for βLG-ASC and βLG-VK3 associations. The results of 3D fluorescence FT-IR and UV-Visible measurements indicated that the binding of above nutrients to βLG may induce conformational and micro-environmental changes of protein. Also, there is a reciprocal complement between spectroscopic techniques and molecular docking modeling. The docking results indicate that the ASC, FOL, and VK3 bind to residues located in the subdomain B of βLG. Finally, this report suggests that βLG could be used as an effective carrier of above nutrients in functional foods.

Diketo modification of curcumin affects its interaction with human serum albumin

NASA Astrophysics Data System (ADS)

Shaikh, Shaukat Ali M.; Singh, Beena G.; Barik, Atanu; Ramani, Modukuri V.; Balaji, Neduri V.; Subbaraju, Gottumukkala V.; Naik, Devidas B.; Indira Priyadarsini, K.

2018-06-01

Curcumin isoxazole (CI) and Curcumin pyrazole (CP), the diketo modified derivatives of Curcumin (CU) are metabolically more stable and are being explored for pharmacological properties. One of the requirements in such activities is their interaction with circulatory proteins like human serum albumin (HSA). To understand this, the interactions of CI and CP with HSA have been investigated employing absorption and fluorescence spectroscopy and the results are compared with that of CU. The respective binding constants of CP, CI and CU with HSA were estimated to be 9.3 × 105, 8.4 × 105 and 2.5 × 105 M-1, which decreased with increasing salt concentration in the medium. The extent of decrease in the binding constant was the highest in CP followed by CI and CU. This revealed that along with hydrophobic interaction other binding modes like electrostatic interactions operate between CP/CI/CU with HSA. Fluorescence quenching studies of HSA with these compounds suggested that both static and dynamic quenching mechanisms operate, where the contribution of static quenching is higher for CP and CI than that for CU. From fluorescence resonance energy transfer studies, the binding site of CU, CI and CP was found to be in domain IIA of HSA. CU was found to bind in closer proximity with Trp214 as compared to CI and CP and the same was responsible for efficient energy transfer and the same was also established by fluorescence anisotropy measurements. Furthermore docking simulation complemented the experimental observation, where both electrostatic as well as hydrophobic interactions were indicated between HSA and CP, CI and CU. This study is useful in designing more stable CU derivatives having suitable binding properties with proteins like HSA.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

PubMed

Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

2014-01-01

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lysosome and Phagosome Stability in Lethal Cell Injury

PubMed Central

Hawkins, Hal K.; Ericsson, Jan L. E.; Biberfeld, Peter; Trump, Benjamin F.

1972-01-01

In two types of cell injury in a tissue culture system, the possibility was tested that lysosome rupture may be a lethal cellular reaction to injury, and thus an important general cause of irreversibility of damage in injured tissue. Prior labeling of secondary lysosomes with the fluorochrome acridine orange, or with ferritin, was used to trace changes in lysosomes after applying an injury. The metabolic inhibitors iodoacetate and cyanide were used together to block the cell's energy supply, or attachment of antiserum and subsequent complement attack were used to damage the surface membrane, producing rapid loss of cell volume control. Living cells were studied by time-lapse phase-contrast cinemicrography and fluorescence microscopy, and samples were fixed at intervals for electron microscopy. The cytolytic action of complement was lethal to sensitized cells within 2 hours, but results showed that lysosomes did not rupture for approximately 4 hours and in fact did not release the fluorescent dye until after reaching the postmortem necrotic phase of injury. Cells treated with metabolic inhibitors also showed irreversible alterations, while lysosomes remained intact and retained the ferritin marker. The fluorochrome marker, acridine orange, escaped from lysosomes early after metabolic injury, but the significance of this observation is not clear. The results are interpreted as evidence against the concept that lysosome rupture threatens the survival of injured cells. The original suicide bag mechanism of cell damage thus is apparently not operative in the systems studied. Lysosomes appear to be relatively stable organelles which, following injury of the types studied, burst only after cell death, acting then as scavengers which help to clear cellular debris. ImagesFigs 5-7Fig 18Fig 19Fig 20Figs 21-23Fig 8Fig 9Fig 10Fig 11Figs 24-27Fig 12Figs 13 and 14Fig 1Fig 2Fig 3Fig 4Fig 15Fig 16Fig 17 PMID:4340333

A New Submersible Imaging-in-flow Instrument to Monitor Nano- and Microplankton: Imaging FlowCytobot

NASA Astrophysics Data System (ADS)

Olson, R. J.; Sosik, H. M.; Shalapyonok, A.

2004-12-01

Understanding of how coastal plankton communities are regulated has traditionally been limited by undersampling, but cabled observatories now provide opportunities to deploy submersible sensors that have high power and data transmission requirements. We have developed an in situ instrument to carry out high-resolution, long term monitoring of phytoplankton and microzooplankton in the size range 10 to100 micrometers, to be deployed at cabled research facilities such as the Martha's Vineyard Coastal Observatory (MVCO). The new instrument is designed to complement FlowCytobot, a submersible flow cytometer currently deployed at MVCO that uses fluorescence and light scattering signals from a laser beam to characterize the smallest phytoplankton cells (less than 10 micrometers). Imaging FlowCytobot uses a combination of flow cytometric and video technology to capture images of organisms for identification and to measure chlorophyll fluorescence associated with each image. Images will be classified using neural net software, while the measurements of chlorophyll fluorescence will allow us to discriminate heterotrophic from phototrophic cells. The new instrument, like the original FlowCytobot is autonomous but remotely programmable. It utilizes a computer controlled syringe pump and distribution valve that allows periodic anti-fouling treatment and analysis of standard beads. Samples are analyzed continuously (0.25 to 2.5 ml per min) and data is sent over a fiber optic link to a remote computer for analysis. Preliminary results indicate that we can detect cells as small as 5 micrometers and discriminate several taxa of diatoms and dinoflagellates.

Distribution of the anticancer drugs doxorubicin, mitoxantrone and topotecan in tumors and normal tissues.

PubMed

Patel, Krupa J; Trédan, Olivier; Tannock, Ian F

2013-07-01

Pharmacokinetic analyses estimate the mean concentration of drug within a given tissue as a function of time, but do not give information about the spatial distribution of drugs within that tissue. Here, we compare the time-dependent spatial distribution of three anticancer drugs within tumors, heart, kidney, liver and brain. Mice bearing various xenografts were treated with doxorubicin, mitoxantrone or topotecan. At various times after injection, tumors and samples of heart, kidney, liver and brain were excised. Within solid tumors, the distribution of doxorubicin, mitoxantrone and topotecan was limited to perivascular regions at 10 min after administration and the distance from blood vessels at which drug intensity fell to half was ~25-75 μm. Although drug distribution improved after 3 and 24 h, there remained a significant decrease in drug fluorescence with increasing distance from tumor blood vessels. Drug distribution was relatively uniform in the heart, kidney and liver with substantially greater perivascular drug uptake than in tumors. There was significantly higher total drug fluorescence in the liver than in tumors after 10 min, 3 and 24 h. Little to no drug fluorescence was observed in the brain. There are marked differences in the spatial distributions of three anticancer drugs within tumor tissue and normal tissues over time, with greater exposure to most normal tissues and limited drug distribution to many cells in tumors. Studies of the spatial distribution of drugs are required to complement pharmacokinetic data in order to better understand and predict drug effects and toxicities.

On the Detectability of Oxygen X-Ray Fluorescence and Its Use as a Solar Photospheric Abundance Diagnostic

NASA Astrophysics Data System (ADS)

Drake, Jeremy J.; Ercolano, Barbara

2008-08-01

Monte Carlo calculations of the O Kα line fluoresced by coronal X-rays and emitted just above the temperature minimum region of the solar atmosphere have been employed to investigate the use of this feature as an abundance diagnostic. While they are quite weak, we estimate line equivalent widths in the range 0.02-0.2 Å, depending on the X-ray plasma temperature. The line remains essentially uncontaminated by blends for coronal temperatures T <= 3 × 106 K and should be quite observable, with a flux gtrsim2 photons s-1 arcmin-2. Model calculations for solar chemical mixtures with an O abundance adjusted up and down by a factor of 2 indicate 35%-60% changes in O Kα line equivalent width, providing a potentially useful O abundance diagnostic. Sensitivity of equivalent width to differences between recently recommended chemical compositions with "high" and "low" complements of the CNO trio important for interpreting helioseismological observations is less acute, amounting to 20%-26% at coronal temperatures T <= 2 × 106 K. While still feasible for discriminating between these two mixtures, uncertainties in measured line equivalent widths and in the models used for interpretation would need to be significantly less than 20%. Provided a sensitive X-ray spectrometer with resolving power >=1000 and suitably well-behaved instrumental profile can be built, X-ray fluorescence presents a viable means for resolving the solar "oxygen crisis."

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

PubMed

Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

2008-07-01

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa

PubMed Central

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-01-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum. PMID:20421194

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

PubMed Central

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

PubMed

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-06-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

Novel genetic tools for diaminopimelic acid selection in virulence studies of Yersinia pestis.

PubMed

Bland, David M; Eisele, Nicholas A; Keleher, Lauren L; Anderson, Paul E; Anderson, Deborah M

2011-03-02

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

PubMed Central

Tait, Brian D.

2016-01-01

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

Nanoscale materials for hyperthermal theranostics

DOE PAGES

Smith, Bennett E.; Roder, Paden B.; Zhou, Xuezhe; ...

2015-03-18

Recently, the use of nanoscale materials has attracted considerable attention with the aim of designing personalized therapeutic approaches that can enhance both spatial and temporal control over drug release, permeability, and uptake. Potential benefits to patients include the reduction of overall drug dosages, enabling the parallel delivery of different pharmaceuticals, and the possibility of enabling additional functionalities such as hyperthermia or deep-tissue imaging (LIF, PET, etc.) that complement and extend the efficacy of traditional chemotherapy and surgery. Our mini review is focused on an emerging class of nanometer-scale materials that can be used both to heat malignant tissue to reducemore » angiogenesis and DNA-repair while simultaneously offering complementary imaging capabilities based on radioemission, optical fluorescence, magnetic resonance, and photoacoustic methods.« less

Cell micro-irradiation with MeV protons counted by an ultra-thin diamond membrane

NASA Astrophysics Data System (ADS)

Barberet, Philippe; Pomorski, Michal; Muggiolu, Giovanna; Torfeh, Eva; Claverie, Gérard; Huss, Cédric; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé

2017-12-01

We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 μm and are used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 μm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

PubMed

Fernández, Aurora Piñas; Gil, Patricia; Valkai, Ildiko; Nagy, Ferenc; Schäfer, Eberhard

2005-05-01

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.

A Unique Collection of Palaeolithic Painted Portable Art: Characterization of Red and Yellow Pigments from the Parpalló Cave (Spain)

PubMed Central

Villaverde Bonilla, Valentín; Ródenas Marín, Isabel; Murcia Mascarós, Sonia

2016-01-01

In this work we analyze the pigments used in the decoration of red and yellow motifs present in the portable art of the Parpalló Cave (Gandía, Spain), one of the most important Palaeolithic sites in the Spanish Mediterranean region. Energy dispersive X-ray fluorescence spectrometry (EDXRF) and spectrophotometry in the visible region (CIEL*a*b*color coordinates and spectral reflectance curves) were used to perform in situ fast analyses of the red and yellow motifs with portable equipment and to characterize their elemental composition and their colorimetric perception, respectively. According to the elemental composition, the intensity of the fluorescence iron signals in red and yellow motifs are higher than average values in the rock substrates. As expected, red motifs possess high values of the chromatic coordinate a* and yellow motifs possess high values of b*. This characterization was complemented with FT-IR analyses of microsamples detached from the red and yellow colored zones of a small set of plaquettes. Our results show that the artists used red and yellow pigments in the decoration likely derived from natural iron oxides as hematite and goethite. PMID:27732605

Cellular uptake and ex vivo urothelial penetration by oligodeoxynucleotides for optimizing treatment of transitional cell carcinoma.

PubMed

Bolenz, Christian; Trojan, Lutz; Gabriel, Ute; Honeck, Patrick; Wendt-Nordahl, Gunnar; Schaaf, Axel; Alken, Peter; Michel, Maurice Stephan

2008-10-01

To evaluate cellular uptake and urothelial penetration of oligodeoxynucleotides (ODNs) in transitional cell carcinoma (TCC) cell lines and in a porcine ex vivo model, respectively. A panel of human TCC cell lines (RT 112, HT 1197 and UM-UC3) were exposed tofluorescein-labeled ODNs. Transfection rates were assessed byfluorescence microscopy and fluorescence-activated cell sorting (FACS). Intravesical treatment with ODNs was performed in a porcine ex vivo model. Urothelial penetration was evaluated using fluorescence microscopy of cryosections. Treatment with ODNs provided transfection rates of at least 96.8% of TCC cells, irrespective of use of a transfection agent. Effective urothelial penetration by ODNs was detected when compared with controls (p = 0.0325). The addition of a liposomal transfection agent significantly increased the penetration depth, allowing affection of deep urothelial cell layers (p = 0.0082). High transfection rates of ODNs can be achieved in TCC cells. Urothelial penetration of ODNs was observed down to the deepest cell layers when a transfection agent is added, suggesting a high potential for complementing the chemoresection effects on residual tumor areas during intravesical therapy of non-muscle-invasive TCC.

Acoustic Sensing and Ultrasonic Drug Delivery in Multimodal Theranostic Capsule Endoscopy

PubMed Central

Stewart, Fraser R.; Qiu, Yongqiang; Newton, Ian P.; Cox, Benjamin F.; Al-Rawhani, Mohammed A.; Beeley, James; Liu, Yangminghao; Huang, Zhihong; Cumming, David R. S.; Näthke, Inke

2017-01-01

Video capsule endoscopy (VCE) is now a clinically accepted diagnostic modality in which miniaturized technology, an on-board power supply and wireless telemetry stand as technological foundations for other capsule endoscopy (CE) devices. However, VCE does not provide therapeutic functionality, and research towards therapeutic CE (TCE) has been limited. In this paper, a route towards viable TCE is proposed, based on multiple CE devices including important acoustic sensing and drug delivery components. In this approach, an initial multimodal diagnostic device with high-frequency quantitative microultrasound that complements video imaging allows surface and subsurface visualization and computer-assisted diagnosis. Using focused ultrasound (US) to mark sites of pathology with exogenous fluorescent agents permits follow-up with another device to provide therapy. This is based on an US-mediated targeted drug delivery system with fluorescence imaging guidance. An additional device may then be utilized for treatment verification and monitoring, exploiting the minimally invasive nature of CE. While such a theranostic patient pathway for gastrointestinal treatment is presently incomplete, the description in this paper of previous research and work under way to realize further components for the proposed pathway suggests it is feasible and provides a framework around which to structure further work. PMID:28671642

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

NASA Astrophysics Data System (ADS)

Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

2005-04-01

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

PubMed Central

Hida, Naoki; Awais, Muhammad; Takeuchi, Masaki; Ueno, Naoto; Tashiro, Mayuri; Takagi, Chiyo; Singh, Tanuja; Hayashi, Makoto; Ohmiya, Yoshihiro; Ozawa, Takeaki

2009-01-01

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. PMID:19536355

A Unique Collection of Palaeolithic Painted Portable Art: Characterization of Red and Yellow Pigments from the Parpalló Cave (Spain).

PubMed

Roldán García, Clodoaldo; Villaverde Bonilla, Valentín; Ródenas Marín, Isabel; Murcia Mascarós, Sonia

2016-01-01

In this work we analyze the pigments used in the decoration of red and yellow motifs present in the portable art of the Parpalló Cave (Gandía, Spain), one of the most important Palaeolithic sites in the Spanish Mediterranean region. Energy dispersive X-ray fluorescence spectrometry (EDXRF) and spectrophotometry in the visible region (CIEL*a*b*color coordinates and spectral reflectance curves) were used to perform in situ fast analyses of the red and yellow motifs with portable equipment and to characterize their elemental composition and their colorimetric perception, respectively. According to the elemental composition, the intensity of the fluorescence iron signals in red and yellow motifs are higher than average values in the rock substrates. As expected, red motifs possess high values of the chromatic coordinate a* and yellow motifs possess high values of b*. This characterization was complemented with FT-IR analyses of microsamples detached from the red and yellow colored zones of a small set of plaquettes. Our results show that the artists used red and yellow pigments in the decoration likely derived from natural iron oxides as hematite and goethite.

Determination of Active Phagocytosis of Unopsonized Porphyromonas gingivalis by Macrophages and Neutrophils Using the pH-Sensitive Fluorescent Dye pHrodo

PubMed Central

Lenzo, Jason C.; O'Brien-Simpson, Neil M.; Cecil, Jessica; Holden, James A.

2016-01-01

Phagocytosis of pathogens is an important component of the innate immune system that is responsible for the removal and degradation of bacteria as well as their presentation via the major histocompatibility complexes to the adaptive immune system. The periodontal pathogen Porphyromonas gingivalis exhibits strain heterogeneity, which may affect a phagocyte's ability to recognize and phagocytose the bacterium. In addition, P. gingivalis is reported to avoid phagocytosis by antibody and complement degradation and by invading phagocytic cells. Previous studies examining phagocytosis have been confounded by both the techniques employed and the potential of the bacteria to invade the cells. In this study, we used a novel, pH-sensitive dye, pHrodo, to label live P. gingivalis strains and examine unopsonized phagocytosis by murine macrophages and neutrophils and human monocytic cells. All host cells examined were able to recognize and phagocytose unopsonized P. gingivalis strains. Macrophages had a preference to phagocytose P. gingivalis strain ATCC 33277 over other strains and clinical isolates in the study, whereas neutrophils favored P. gingivalis W50, ATCC 33277, and one clinical isolate over the other strains. This study revealed that all P. gingivalis strains were capable of being phagocytosed without prior opsonization with antibody or complement. PMID:27021243

Inactivation of a subpopulation of human neutrophils by exposure to ultrahigh-molecular-weight polyethylene wear debris.

PubMed

Bernard, Louis; Vaudaux, Pierre; Huggler, Elzbieta; Stern, Richard; Fréhel, Claude; Francois, Patrice; Lew, Daniel; Hoffmeyer, Pierre

2007-04-01

Polymorphonuclear neutrophils, a first line of defence against invading microbial pathogens, may be attracted by inflammatory mediators triggered by ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles released from orthopaedic prostheses. Phagocytosis of UHMWPE particles by neutrophils may indirectly compromise their phagocytic-bactericidal mechanisms, thus enhancing host susceptibility to microbial infections. In an in vitro assay, pre-exposure of purified human neutrophils to UHMWPE micrometre- and submicrometre-sized wear particles interfered with subsequent Staphylococcos aureus uptake in a heterogeneous way, as assessed by a dual label fluorescence microscopic assay that discriminated intracellular rhodamine-labelled UHMWPE particles from fluorescein isothiocyanate-labelled S. aureus. Indeed, a higher percentage (44%) of neutrophils having engulfed UHMWPE particles lost the ability to phagocytize S. aureus, compared with UHMWPE-free neutrophils (<3%). Pre-exposure of neutrophils to UHMWPE wear particles did not impair but rather stimulated their oxidative burst response in a chemoluminescence assay. The presence of UHMWPE wear particles did not lead to significant overall consumption of complement-mediated opsonic factors nor decreased surface membrane display of neutrophil complement receptors. In conclusion, engulfment of UHMWPE wear particles led to inactivation of S. aureus uptake in nearly half of the neutrophil population, which may potentially impair host clearance mechanisms against pyogenic infections.

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

PubMed Central

Shell, Steven M.; Hawkins, Edward K.; Tsai, Miaw-Sheue; Hlaing, Aye Su; Rizzo, Carmelo J.; Chazin, Walter J.

2013-01-01

The xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results support an indirect read-out model for sensing the presence of lesions by human XPC and suggest XPC may act as a general sensor of damaged DNA capable of recognizing DNA containing lesions not repaired by NER. PMID:24051049

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Measurements of Primary Biogenic Aerosol Particles with an Ultraviolet Aerodynamic Particle Sizer (UVAPS) During AMAZE-08

NASA Astrophysics Data System (ADS)

Wollny, A. G.; Garland, R.; Pöschl, U.

2008-12-01

Biogenic aerosols are ubiquitous in the Earth's atmosphere and they influence atmospheric chemistry and physics, the biosphere, climate, and public health. They play an important role in the spread of biological organisms and reproductive materials, and they can cause or enhance human, animal, and plant diseases. Moreover, they influence the Earth's energy budget by scattering and absorbing radiation, and they can initiate the formation of clouds and precipitation as cloud condensation and ice nuclei. The composition, abundance, and origin of biogenic aerosol particles and components are, however, still not well understood and poorly quantified. Prominent examples of primary biogenic aerosol particles, which are directly emitted from the biosphere to the atmosphere, are pollen, bacteria, fungal spores, viruses, and fragments of animals and plants. During the AMazonian Aerosol CharacteriZation Experiment (AMAZE-08) a large number of aerosol and gas-phase measurements were taken on a remote site close to Manaus, Brazil, during a period of five weeks in February and March 2008. The presented study is focused on data from an ultraviolet aerodynamic particle sizer (UVAPS, TSI inc.) that has been deployed for the first time in Amazonia. In this instrument, particle counting and aerodynamic sizing over the range of 0.5-20 μm are complemented by the measurement of UV fluorescence at 355 nm (excitation) and 420-575 nm (emission), respectively. Fluorescence at these wavelengths is characteristic for reduced pyridine nucleotides (e.g., NAD(P)H) and for riboflavin, which are specific for living cells. Thus particles exhibiting fluorescence signals can be regarded as 'viable aerosols' or 'fluorescent bioparticles' (FBAP), and their concentration can be considered as lower limit for the actual abundance of primary biogenic aerosol particles. First data analyses show a pronounced peak of FBAP at diameters around 2-3 μm. In this size range the biogenic particle fraction was generally higher than 50 %. Additionally, bursts of FBAP have been observed nearly every day just before sunrise. During these periods the coarse (super-micron) aerosol consisted almost completely of fluorescent bioparticles.

Time Resolved Measurements of Primary Biogenic Aerosol Particles in Amazonia

NASA Astrophysics Data System (ADS)

Wollny, A. G.; Garland, R.; Pöschl, U.

2009-04-01

Biogenic aerosols are ubiquitous in the Earth's atmosphere and they influence atmospheric chemistry and physics, the biosphere, climate, and public health. They play an important role in the spread of biological organisms and reproductive materials, and they can cause or enhance human, animal, and plant diseases. Moreover, they influence the Earth's energy budget by scattering and absorbing radiation, and they can initiate the formation of clouds and precipitation as cloud condensation and ice nuclei. The composition, abundance, and origin of biogenic aerosol particles and components are, however, still not well understood and poorly quantified. Prominent examples of primary biogenic aerosol particles, which are directly emitted from the biosphere to the atmosphere, are pollen, bacteria, fungal spores, viruses, and fragments of animals and plants. During the Amazonian Aerosol Characterization Experiment (AMAZE-08) a large number of aerosol and gas-phase measurements were taken on a remote site close to Manaus, Brazil, during a period of five weeks in February and March 2008. This presented study is focused on data from an ultraviolet aerodynamic particle sizer (UVAPS, TSI inc.) that has been deployed for the first time in Amazonia. In this instrument, particle counting and aerodynamic sizing over the range of 0.5-20 µm are complemented by the measurement of UV fluorescence at 355 nm (excitation) and 420-575 nm (emission), respectively. Fluorescence at these wavelengths is characteristic for reduced pyridine nucleotides (e.g., NAD(P)H) and for riboflavin, which are specific for living cells. Thus particles exhibiting fluorescence signals can be regarded as "viable aerosols" or "fluorescent bioparticles" (FBAP), and their concentration can be considered as lower limit for the actual abundance of primary biogenic aerosol particles. Data from the UVAPS were averaged over 5 minute time intervals. The presence of bioparticles in the observed size range has been confirmed by filter samples. First data analyses show a pronounced peak of FBAP at diameters around 2-3 µm. In this size range the biogenic particle fraction was generally higher than 50%. Additionally, bursts of FBAP have been observed nearly every day just before sunrise. During these periods the coarse (super-micron) aerosol consisted almost completely out of fluorescent bioparticles.

Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

PubMed

Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

2014-02-01

The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the nucleus at high concentration. Exposure to low concentrations of cobalt or silver did not alter the localization nor the concentration of endogenous elements within the cells. To our knowledge, this is the first report on element co-localization and segregation at the sub-cellular level in micro-algae by means of synchrotron nano X-ray fluorescence spectroscopy.

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

PubMed

Dantas de Araujo, Aline; Wu, Chongyang; Wu, Kai-Chen; Reid, Robert C; Durek, Thomas; Lim, Junxian; Fairlie, David P

2017-06-21

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (K d ) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pK i of 8.6 ± 0.2 and K i of 2.5 nM) and C3aR ligands TR16 (pK i of 6.8 ± 0.1 and K i of 138 nM), BR103 (pK i of 6.7 ± 0.1 and K i of 185 nM), BR111 (pK i of 6.3 ± 0.2 and K i of 544 nM) and SB290157 (pK i of 6.3 ± 0.1 and K i of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants.

PubMed

Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

2015-09-11

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

Use of PMA1 as a Housekeeping Biomarker for Assessment of Toxicant-Induced Stress in Saccharomyces cerevisiae

PubMed Central

Schmitt, Marcel; Schwanewilm, Petra; Ludwig, Jost; Lichtenberg-Fraté, Hella

2006-01-01

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence. The reporter assay deployed a simple and robust protocol for the rapid detection of toxic effects within a 96-well microplate format. Fluorescence emissions were normalized to cell growth determined by absorption and were correlated to internal reference standards. The results were expressed as effective concentrations (EC20). Dose-response experiments were conducted in which yeast cells were exposed in minimal medium and in the presence of 20% fetal calf serum to sublethal concentrations of an array of heavy metals, salt, and a number of stress-inducing compounds (Diclofenac, Lindane, methyl-N-nitro-N-nitrosoguanidine [MNNG], hydroxyurea, and caffeine). Long-term exposure (7 h) played a considerable role in the adaptive response to intoxication compared to early responses at 4 h exposure. The data obtained after 4 h of exposure and expressed as EC20 were compared to 50% inhibitory concentration values derived from cell line and ecotoxicological tests. This study demonstrates the versatility of the novel biomarker to complement existing test batteries to assess contaminant exposure and effects. PMID:16461706

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Arabidopsis G-protein β subunit AGB1 interacts with NPH3 and is involved in phototropism.

PubMed

Kansup, Jeeraporn; Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

2014-02-28

Heterotrimeric G proteins (Gα, Gβ and Gγ) have pleiotropic roles in plants, but molecular mechanisms underlying them remain to be elucidated. Here we show that Arabidopsis Gβ (AGB1) interacts with NPH3, a regulator of phototropism. Yeast two-hybrid assays, in vitro pull-down assays and bimolecular fluorescence complementation assays showed that AGB1 and NPH3 physically interact. NPH3-null mutation (nph3) is known to completely abolish hypocotyl phototropism. Loss-of-function mutants of AGB1 (agb1-1 and agb1-2) showed decreased hypocotyl phototropism, and agb1/nph3 double mutants showed no hypocotyl phototropism. These results suggest that AGB1 is involved in the NPH3-mediated regulation of phototropism. Copyright © 2014 Elsevier Inc. All rights reserved.

PubMed Central

Montplaisir, S.; Côté, P. P.; Martineau, B.; Roche, A. J.; Mongeau, J. G.; Robitaille, P.

1976-01-01

The demonstration by immuno-fluorescence of antibodies on the surface of urinary bacteria, a new method of determining the site of a urinary tract infection, was found to be as valuable in children as it is in adults. A clear correlation exists between a positive test result and renal parenchymal infection on one hand, and a negative result and lower urinary tract infection on the other. Moreover, immunoglobulins were still detectable in original positive urine samples that had been standing at 4degrees C for 7 weeks. The constant finding of IgA on bacteria suggests a particular synthesis for this class of immunoglobulin. A pathophysiologic role for complement would appear to be excluded by the facts that the serum concentrations of C3 were normal and that C3 was invariably absent from the bacterial surface. Images FIG. 1 PMID:793701

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy

PubMed Central

Small, David M.; Jones, Jason S.; Tendler, Irwin I.; Miller, Paul E.; Ghetti, Andre; Nishimura, Nozomi

2017-01-01

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool. PMID:29359098

Daily behavior of urban Fluorescing Aerosol Particles in northwest Spain

NASA Astrophysics Data System (ADS)

Calvo, A. I.; Baumgardner, D.; Castro, A.; Fernández-González, D.; Vega-Maray, A. M.; Valencia-Barrera, R. M.; Oduber, F.; Blanco-Alegre, C.; Fraile, R.

2018-07-01

Measurements of ambient aerosol particles at the University of León, León, Spain, were made in May and June 2015 with a Wideband Integrated Bioaerosol Spectrometer (WIBS). The WIBS detects Fluorescing Aerosol Particles (FAP) in the size range from 0.5 to 20 μm. These measurements were complemented with an analysis of pollen concentrations assessed with optical microscopy of samples captured with a volumetric Hirst spore trap. The total particle, FAP and pollen concentrations show clear, daily cycles. Whereas the total particle concentrations maximize at 0800 and 2200 UTC, the FAP concentrations have peaks at midnight and 0800 UTC while the pollen has a broad peak between 1200 and 2000 UTC. The FAP larger than 2 μm represent 15-35% of the total particle population in this size range, maximizing at midnight UTC. Similar to what has been found by investigators at other locations, there is a strong positive correlation of the WIBS measured FAP with relative humidity; however, the pollen concentration is positively correlated with the temperature and anti-correlated with the relative humidity. Back trajectory analysis indicates that the largest FAP to total particle fractions are found in air masses arriving from the northeast with the second largest coming from the southwest. Given the location of the university in relation to the city and forested areas, this implies that the higher concentration FAP are coming from rural, probably natural, sources; however, more local, anthropogenic sources cannot be ruled out as a secondary source. The majority of the FAP that are identified from microscopy are fungal spores (Cladosporium, Aspergillus, Alternaria, Oidium) and pollen grains (mainly Poaceae, Quercus, Plantago, Rumex and Urticaceae). A comparison of the fluorescence fingerprints between laboratory generated FAP and the ambient particles showed some similarities; however, a significant fraction of the FAP are those whose fluorescence patterns do not match any of those that have been previously classified in the laboratory.

Time-resolved confocal fluorescence microscopy: novel technical features and applications for FLIM, FRET and FCS using a sophisticated data acquisition concept in TCSPC

NASA Astrophysics Data System (ADS)

Koberling, Felix; Krämer, Benedikt; Kapusta, Peter; Patting, Matthias; Wahl, Michael; Erdmann, Rainer

2007-05-01

In recent years time-resolved fluorescence measurement and analysis techniques became a standard in single molecule microscopy. However, considering the equipment and experimental implementation they are typically still an add-on and offer only limited possibilities to study the mutual dependencies with common intensity and spectral information. In contrast, we are using a specially designed instrument with an unrestricted photon data acquisition approach which allows to store spatial, temporal, spectral and intensity information in a generalized format preserving the full experimental information. This format allows us not only to easily study dependencies between various fluorescence parameters but also to use, for example, the photon arrival time for sorting and weighting the detected photons to improve the significance in common FCS and FRET analysis schemes. The power of this approach will be demonstrated for different techniques: In FCS experiments the concentration determination accuracy can be easily improved by a simple time-gated photon analysis to suppress the fast decaying background signal. A more detailed analysis of the arrival times allows even to separate FCS curves for species which differ in their fluorescence lifetime but, for example, cannot be distinguished spectrally. In multichromophoric systems like a photonic wire which undergoes unidirectional multistep FRET the lifetime information complements significantly the intensity based analysis and helps to assign the respective FRET partners. Moreover, together with pulsed excitation the time-correlated analysis enables directly to take advantage of alternating multi-colour laser excitation. This pulsed interleaved excitation (PIE) can be used to identify and rule out inactive FRET molecules which cause interfering artefacts in standard FRET efficiency analysis. We used a piezo scanner based confocal microscope with compact picosecond diode lasers as excitation sources. The timing performance can be significantly increased by using new SPAD detectors which enable, in conjunction with new TCSPC electronics, an overall IRF width of less than 120 ps maintaining single molecule sensitivity.

Effects of hepatitis B virus infection on human sperm chromosomes.

PubMed

Huang, Jian-Min; Huang, Tian-Hua; Qiu, Huan-Ying; Fang, Xiao-Wu; Zhuang, Tian-Gang; Liu, Hong-Xi; Wang, Yong-Hua; Deng, Li-Zhi; Qiu, Jie-Wen

2003-04-01

To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients. Sperm chromosomes of 14 tested subjects (5 healthy controls, 9 patients with HBV infection, including 1 with acute hepatitis B, 2 with chronic active hepatitis B, 4 with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. The total frequency of sperm chromosome aberrations in HBV infection group (14.8 %, 33/223) was significantly higher than that in the control group (4.3 %, 5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4 signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random. HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Structural and biophysical properties of h-FANCI ARM repeat protein.

PubMed

Siddiqui, Mohd Quadir; Choudhary, Rajan Kumar; Thapa, Pankaj; Kulkarni, Neha; Rajpurohit, Yogendra S; Misra, Hari S; Gadewal, Nikhil; Kumar, Satish; Hasan, Syed K; Varma, Ashok K

2017-11-01

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

PubMed Central

Binder, Andreas; Lambert, Jayne; Morbitzer, Robert; Popp, Claudia; Ott, Thomas; Lahaye, Thomas; Parniske, Martin

2014-01-01

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct. PMID:24551083

Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62.

PubMed

Nibe, Yoichi; Oshima, Shigeru; Kobayashi, Masanori; Maeyashiki, Chiaki; Matsuzawa, Yu; Otsubo, Kana; Matsuda, Hiroki; Aonuma, Emi; Nemoto, Yasuhiro; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Nakada, Shinichiro; Watanabe, Mamoru

2018-01-01

Ubiquitin chains are formed with 8 structurally and functionally distinct polymers. However, the functions of each polyubiquitin remain poorly understood. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay using Kusabira Green (KG) as a split fluorescent protein. The PolyUb-FC assay has the advantage that monoubiquitination is nonfluorescent and chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine K33-linked polyubiquitin. We demonstrated that SQSTM1/p62 puncta colocalized with K33-linked polyubiquitin and this interaction was modulated by the ZRANB1/TRABID-K29 and -K33 linkage-specific deubiquitinase (DUB). We further showed that the colocalization of K33-linked polyubiquitin and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) puncta was impaired by SQSTM1/p62 deficiency. Taken together, these findings provide novel insights into how atypical polyubiquitin is recruited by SQSTM1/p62. Finally, we developed an inducible-PolyUb-FC system for visualizing chain-specific polyubiquitin. The PolyUb-FC will be a useful tool for analyzing the dynamics of atypical polyubiquitin chain generation.

Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

PubMed

Bereza-Malcolm, Lara; Aracic, Sanja; Kannan, Ruban; Mann, Gülay; Franks, Ashley E

2017-08-15

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml -1 , as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml -1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera. Copyright © 2017 Elsevier B.V. All rights reserved.

A Photo-triggered and photo-calibrated nitric oxide donor: Rational design, spectral characterizations, and biological applications.

PubMed

He, Haihong; Liu, Yuxin; Zhou, Zhongneng; Guo, Chunlei; Wang, Hong-Yin; Wang, Zhuang; Wang, Xueli; Zhang, Ziqian; Wu, Fu-Gen; Wang, Haolu; Chen, Daijie; Yang, Dahai; Liang, Xiaowen; Chen, Jinquan; Zhou, Shengmin; Liang, Xin; Qian, Xuhong; Yang, Youjun

2018-04-27

Nitric oxide (NO) donors are valuable tools to probe the profound implications of NO in health and disease. The elusive nature of NO bio-relevance has largely limited the use of spontaneous NO donors and promoted the development of next generation NO donors, whose NO release is not only stimulated by a trigger, but also readily monitored via a judiciously built-in self-calibration mechanism. Light is without a doubt the most sensitive, versatile and biocompatible method of choice for both triggering and monitoring, for applications in complex biological matrices. Herein, we designed and synthesized an N-nitroso rhodamine derivative (NOD560) as a photo-triggered and photo-calibrated NO donor to address this need. NOD560 is essentially non-fluorescent. Upon irradiation by green light (532 nm), it efficiently release NO and a rhodamine dye, the dramatic fluorescence turn-on from which could be harnessed to conveniently monitor the localization, flux, and dose of NO release. The potentials of NOD560 for in vitro biological applications were also exemplified in in vitro biological models, i.e. mesenchymal stem cell (MSC) migration suppression. NOD560 is expected to complement the existing NO donors and find widespread applications in chemical biological studies. Copyright © 2018 Elsevier Inc. All rights reserved.

Arraycount, an algorithm for automatic cell counting in microwell arrays.

PubMed

Kachouie, Nezamoddin; Kang, Lifeng; Khademhosseini, Ali

2009-09-01

Microscale technologies have emerged as a powerful tool for studying and manipulating biological systems and miniaturizing experiments. However, the lack of software complementing these techniques has made it difficult to apply them for many high-throughput experiments. This work establishes Arraycount, an approach to automatically count cells in microwell arrays. The procedure consists of fluorescent microscope imaging of cells that are seeded in microwells of a microarray system and then analyzing images via computer to recognize the array and count cells inside each microwell. To start counting, green and red fluorescent images (representing live and dead cells, respectively) are extracted from the original image and processed separately. A template-matching algorithm is proposed in which pre-defined well and cell templates are matched against the red and green images to locate microwells and cells. Subsequently, local maxima in the correlation maps are determined and local maxima maps are thresholded. At the end, the software records the cell counts for each detected microwell on the original image in high-throughput. The automated counting was shown to be accurate compared with manual counting, with a difference of approximately 1-2 cells per microwell: based on cell concentration, the absolute difference between manual and automatic counting measurements was 2.5-13%.

A secretory multifunctional serine protease, DegP of Plasmodium falciparum, plays an important role in thermo-oxidative stress, parasite growth and development.

PubMed

Sharma, Shweta; Jadli, Mohit; Singh, Anu; Arora, Kavita; Malhotra, Pawan

2014-03-01

Plasmodium falciparum heat shock proteins and proteases are known for their indispensable roles in parasite virulence and survival in the host cell. They neutralize various host-derived stress responses that are deleterious for parasite growth and invasion. We report identification and functional characterization of the first DegP from an apicomplexan (P. falciparum). To determine the molecular identity and functions of the parasite-encoded DegP, we complemented the Escherichia coli degP null mutant with a putative PfdegP gene, and the results showed that PfDegP complements the growth defect of the temperature sensitive DegP-deficient mutant and imparts resistance to non-permissive temperatures and oxidative stress. Molecular interaction studies showed that PfDegP exists as a complex with parasite-encoded heat shock protein 70, iron superoxide dismutase and enolase. DegP expression is significantly induced in parasite culture upon heat shock/oxidative stress. Our data suggest that the PfDegP protein may play a role in the growth and development of P. falciparum through its ability to confer protection against thermal/oxidative stress. Antibody against DegP showed anti-plasmodial activity against blood-stage parasites in vitro, suggesting that PfDegP and its associated complex may be a potential focus for new anti-malarial therapies. ●PfDegP physically interacts with PfHsp70 and PfEno by anti-bait co-immunoprecipitation (View interaction) ●PfDegP physically interacts with PfEno, PfSod, PfOat, PfHsp70, PfLDH and PfGpi by anti-bait co-immunoprecipitation (View interaction) ●PfHsp-70 and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP physically interacts with PfOat, PfHsp70, PfEno, PfSod, PfGpi and PfLDH by surface plasmon resonance (View interaction) ●PfEno and PfDegP co-localize by fluorescence microscopy (View interaction) ●PfDegP and PfHsp70 co-localize by co-sedimentation through density gradient (View interaction). © 2014 FEBS.

The Ustilago maydis Effector Pep1 Suppresses Plant Immunity by Inhibition of Host Peroxidase Activity

PubMed Central

Zechmann, Bernd; Hillmer, Morten; Doehlemann, Gunther

2012-01-01

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction. PMID:22589719

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

PubMed

Becker, P S; Taylor, J A; Trobridge, G D; Zhao, X; Beard, B C; Chien, S; Adair, J; Kohn, D B; Wagner, J E; Shimamura, A; Kiem, H-P

2010-10-01

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes

PubMed Central

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction. PMID:29385206

[Localization and composition of renal immunodeposits in mice developing HgCl2-induced autoimmune process].

PubMed

Aref'eva, A S; Dyban, P A; Krasil'shchikova, M S; Dobrucki, J W; Zatsepina, O V

2010-01-01

A characteristic feature of systemic autoimmune diseases along with appearance of autoantibodies targeting self-antigenes is deposition of immunoglobulins and components of the complement system in kidneys. However, mechanisms of the deposit formation and their cytotoxic effects still remain poorly studied. To elucidate these questions, we used SJL/J mice which are known to develop autoimmune process accompanied by the appearance of anti-fibrillarin antibodies following regular administrations of sublethal dozes of HgCl2. Using antibodies to the total murine ummunoglobulins we showed that immunodeposits were present in glomeruli of autoimmune and control (not-autoimmune) animals, but their intensity was directly correlated with the titer of anti-fibrillarin autoantibodies and was minimal in control mice. By confocal microscopy and conventional fluorescence microscopy it was defined that immunodeposits deeply penetrate glomeruli and are the most likely located within mesangial cells. In autoimmune animals, ummunoglobulins completely colocolized with the C3--component of complement, but not with the major autoantigen--the protein fibrillarin. We failed to determine the signs of cell proliferation or death in glomeruli. The most prominent difference between control and autoimmune mice was the presence if immunodeposits in renal blood vessels. These observations argue in favor of the idea that destructive and disfunctional renal lesions accompanying development of autoimmune diseases can be caused, in part, by accumulation of immunodeposits in blood vessels.

The NB-LRR gene Pm60 confers powdery mildew resistance in wheat.

PubMed

Zou, Shenghao; Wang, Huan; Li, Yiwen; Kong, Zhaosheng; Tang, Dingzhong

2018-04-01

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucine-rich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiled-coil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ †

PubMed Central

Laligné, C.; Klotz, C.; Garreau de Loubresse, N.; Lemullois, M.; Hori, M.; Laurent, F. X.; Papon, J. F.; Louis, B.; Cohen, J.; Koll, F.

2010-01-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate. PMID:20118210

Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

PubMed

Laligné, C; Klotz, C; de Loubresse, N Garreau; Lemullois, M; Hori, M; Laurent, F X; Papon, J F; Louis, B; Cohen, J; Koll, F

2010-04-01

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes.

PubMed

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh; Lood, Rolf

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts[W

PubMed Central

Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.

2010-01-01

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559

The Na+-phosphate cotransport system (NaPi-II) with a cleaved protein backbone: implications on function and membrane insertion

PubMed Central

Kohl, Beate; Wagner, Carsten A; Huelseweh, Birgit; Busch, Andreas E; Werner, Andreas

1998-01-01

Renal handling of inorganic phosphate (Pi) involves a Na+-Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi-II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N- (1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaPi-II from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased Pi flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaPi-II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) was lower. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence. PMID:9508800

Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

PubMed

Singer, John T; Phennicie, Ryan T; Sullivan, Matthew J; Porter, Laura A; Shaffer, Valerie J; Kim, Carol H

2010-06-01

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

PubMed Central

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

PubMed

Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

2018-05-11

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

MdHB1 down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar 'Granny Smith'.

PubMed

Jiang, Yonghua; Liu, Cuihua; Yan, Dan; Wen, Xiaohong; Liu, Yanli; Wang, Haojie; Dai, Jieyu; Zhang, Yujie; Liu, Yanfei; Zhou, Bin; Ren, Xiaolin

2017-02-01

Coloration in apple (Malus×domestica) flesh is mainly caused by the accumulation of anthocyanin. Anthocyanin is biosynthesized through the flavonoid pathway and regulated by MYB, bHLH, and WD40 transcription factors (TFs). Here, we report that the HD-Zip I TF MdHB1 was also involved in the regulation of anthocyanin accumulation. MdHB1 silencing caused the accumulation of anthocyanin in 'Granny Smith' flesh, whereas its overexpression reduced the flesh content of anthocyanin in 'Ballerina' (red-fleshed apple). Moreover, flowers of transgenic tobacco (Nicotiana tabacum 'NC89') overexpressing MdHB1 showed a remarkable reduction in pigmentation. Transient promoter activation assays and yeast one-hybrid results indicated that MdHB1 indirectly inhibited expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and UDP-glucose:flavonoid 3-O-glycosyltransferase (UFGT). Yeast two-hybrid and bimolecular fluorescence complementation determined that MdHB1 acted as a homodimer and could interact with MYB, bHLH, and WD40 in the cytoplasm, consistent with its cytoplasmic localization by green fluorescent protein fluorescence observations. Together, these results suggest that MdHB1 constrains MdMYB10, MdbHLH3, and MdTTG1 to the cytoplasm, and then represses the transcription of MdDFR and MdUFGT indirectly. When MdHB1 is silenced, these TFs are released to activate the expression of MdDFR and MdUFGT and also anthocyanin biosynthesis, resulting in red flesh in 'Granny Smith'. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

An open label trial of folate receptor-targeted intraoperative molecular imaging to localize pulmonary squamous cell carcinomas

PubMed Central

Predina, Jarrod D.; Newton, Andrew D.; Xia, Leilei; Corbett, Christopher; Connolly, Courtney; Shin, Michael; Sulyok, Lydia Frezel; Litzky, Leslie; Deshpande, Charuhas; Nie, Shuming; Kularatne, Sumith A.; Low, Philip S.; Singhal, Sunil

2018-01-01

Background Clinical applicability of folate receptor-targeted intraoperative molecular imaging (FR-IMI) has been established for surgically resectable pulmonary adenocarcinoma. A role for FR-IMI in other lung cancer histologies has not been studied. In this study, we evaluate feasibility of FR-IMI in patients undergoing pulmonary resection for squamous cell carcinomas (SCCs). Methods In a human clinical trial (NCT02602119), twelve subjects with pulmonary SCCs underwent FR-IMI with a near-infrared contrast agent that targets the folate receptor-α (FRα), OTL38. Near-infrared signal from tumors and benign lung was quantified to calculate tumor-to-background ratios (TBR). Folate receptor-alpha expression was characterized, and histopathologic correlative analyses were performed to evaluate patterns of OTL38 accumulation. An exploratory analysis was performed to determine patient and histopathologic variables that predict tumor fluorescence. Results 9 of 13 SCCs (in 9 of 12 of subjects) displayed intraoperative fluorescence upon NIR evaluation (median TBR, 3.9). OTL38 accumulated within SCCs in a FRα-dependent manner. FR-IMI was reliable in localizing nodules as small as 1.1 cm, and prevented conversion to thoracotomy for nodule localization in three subjects. Upon evaluation of patient and histopathologic variables, in situ fluorescence was associated with distance from the pleural surface, and was independent of alternative variables including tumor size and metabolic activity. Conclusions This work demonstrates that FR-IMI is potentially feasible in 70% of SCC patients, and that molecular imaging can improve localization during minimally invasive pulmonary resection. These findings complement previous data demonstrating that ∼98% of pulmonary adenocarcinomas are localized during FR-IMI and suggest broad applicability for NSCLC patients undergoing resection. PMID:29568374

Single cell analysis using surface enhanced Raman scattering (SERS) tags

PubMed Central

Nolan, John P.; Duggan, Erika; Liu, Er; Condello, Danilo; Dave, Isha; Stoner, Samuel A.

2013-01-01

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis. PMID:22498143

Optically-Enhanced, Near-IR, Silver Cluster Emission Altered by Single Base Changes in the DNA Template

PubMed Central

Fan, Chaoyang; Story, Sandra P.; Sengupta, Bidisha; Sartin, Matthew; Hsiang, Jung-Cheng; Perry, Joseph W.

2011-01-01

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near infrared-emitting clusters. Clusters emitting at ~800 nm form with C3AC3AC3TC3A and C3AC3AC3GC3A and both exhibit a trap state with λmax ~ 840 nm and an absorption cross section of 5–6 × 10−16 cm2/molec that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3–4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ~2 × 10−18 cm2/molec. Relative to C3AC3AC3TC3A, C3AC3AC3GC3A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C3AC3AC3AC3G template, a spectrally distinct cluster forms having emission at ~900 nm and its trap state has a ~four-fold shorter lifetime. These studies of optically-gated fluorescence bolster the critical role of the nucleobases on both the formation and excited state dynamics of these highly emissive metallic clusters. PMID:21568292

SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

PubMed

Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

2011-02-04

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants1[OPEN

PubMed Central

Hartings, Stephanie; Paradies, Susanne; Karnuth, Bianca; Eisfeld, Sabrina; Mehsing, Jasmin; Wolff, Christian; Levey, Tatjana

2017-01-01

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 (hcf222-1) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1, the accumulation of the cytochrome b6f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis (Arabidopsis thaliana) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane. PMID:28572458

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants.

PubMed

Hartings, Stephanie; Paradies, Susanne; Karnuth, Bianca; Eisfeld, Sabrina; Mehsing, Jasmin; Wolff, Christian; Levey, Tatjana; Westhoff, Peter; Meierhoff, Karin

2017-07-01

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 ( hcf222-1 ) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1 , the accumulation of the cytochrome b 6 f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis ( Arabidopsis thaliana ) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane. © 2017 American Society of Plant Biologists. All Rights Reserved.

Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy.

PubMed

Sarrion-Perdigones, Alejandro; Palaci, Jorge; Granell, Antonio; Orzaez, Diego

2014-01-01

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction-ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named "GB parts" and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4-5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.

Angular Distribution of light emission in ELVES events

NASA Astrophysics Data System (ADS)

Mussa, Roberto

2017-04-01

The Pierre Auger Observatory, located in Malargüe (Argentina), is the largest facility (3000 kmq ) for the study of Ultra High Energy Cosmic Rays (E>0.3 EeV). The four sites of the Fluorescence Detector (FD) are continuously observing the night sky with moon fraction below 50% (13% duty cycle) with 100 ns time resolution and a space resolution below one degree. Since 2013, the Observatory has implemented a dedicated trigger for the study of ELVES events, produced by lightning activity in Northern Argentina during summer months. A network of ancillary devices (lidars, cloud cameras, weather stations, lightning sensors, E-field mills) complements the FD data to correct for the variation of atmospheric optical properties. This paper will report about the angular distribution of the light emission around the vertical above the lightning source and compare with existing models.

Synthesis and evaluation of di- and trimeric hydroxylamine-based β-(1→3)-glucan mimetics.

PubMed

Ferry, Angélique; Malik, Gaëlle; Guinchard, Xavier; Vĕtvička, Václav; Crich, David

2014-10-22

Di- and trimeric hydroxylamine-based mimetics of β-(1→3)-glucans have been accessed by an asymmetric synthesis route featuring an iterative double ring-closing reductive amination reaction. These oligomeric hydroxylamines are demonstrated to inhibit the staining of human neutrophils and of mouse macrophages by fluorescent anti-CR3 and anti-dectin-1 antibodies, respectively, and to stimulate phagocytosis, all in a linkage-dependent manner suggestive of binding to the lectin domains of complement receptor 3 (CR3) and dectin-1. The ability of these relatively short mimetics to bind to CR3 and dectin-1, as compared to the greater degree of polymerization required in β-(1→3)-glucans, is discussed in terms of the increased hydrophobicity of the α-face on replacement of the glycosidic bond by the hydroxylamine linkage.

Laser-based standoff detection of explosives: a critical review.

PubMed

Wallin, Sara; Pettersson, Anna; Ostmark, Henric; Hobro, Alison

2009-09-01

A review of standoff detection technologies for explosives has been made. The review is focused on trace detection methods (methods aiming to detect traces from handling explosives or the vapours surrounding an explosive charge due to the vapour pressure of the explosive) rather than bulk detection methods (methods aiming to detect the bulk explosive charge). The requirements for standoff detection technologies are discussed. The technologies discussed are mostly laser-based trace detection technologies, such as laser-induced-breakdown spectroscopy, Raman spectroscopy, laser-induced-fluorescence spectroscopy and IR spectroscopy but the bulk detection technologies millimetre wave imaging and terahertz spectroscopy are also discussed as a complement to the laser-based methods. The review includes novel techniques, not yet tested in realistic environments, more mature technologies which have been tested outdoors in realistic environments as well as the most mature millimetre wave imaging technique.

Characterization of Three Carbon- and Nitrogen-Rich Particles from Comet 81P/WILD

NASA Technical Reports Server (NTRS)

Gallien, J.-P.; Khodja, H.; Herzog, G. F.; Taylor, S.; Koepsell, E.; Daghlian, C. P.; Flynn, G. J.; Sitnitsky, I.; Lanzirotti, A.; Sutton, S. R.;

Validation of a multi-analyte panel with cell-bound complement activation products for systemic lupus erythematosus.

PubMed

Dervieux, Thierry; Conklin, John; Ligayon, Jo-Anne; Wolover, Leilani; O'Malley, Tyler; Alexander, Roberta Vezza; Weinstein, Arthur; Ibarra, Claudia A

2017-07-01

We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies. The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays. Results of the multi-analyte panel are reported as positive or negative based on a 2-tiered index score. Post-phlebotomy stability of EC4d and BC4d in EDTA-anticoagulated blood is determined using specimens collected from patients with SLE and normal donors. Three-level C4 coated positive beads are run daily as controls. Analytical validity is reported using intra-day and inter-day coefficient of variation (CV). EC4d and BC4d are stable for 2days at ambient temperature and for 4days at 4°C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n=30) and 7.3% to 12.4% (n=66), respectively. The 2-tiered index score is reproducible over 4 consecutive daysupon storage of blood at 4°C. A total of 2,888 three-level quality control data were collected from 6 flow cytometers with an overall failure rate below 3%. Median EC4d level is 6 net MFI (Interquartile [IQ] range 4-9 net MFI) and median BC4d is 18 net MFI (IQ range 13-27 net MFI) among 86,852 specimens submitted for testing. The incidence of 2-tiered positive test results is 13.4%. We have established the analytical validity of a multi-analyte assay panel for SLE. Copyright © 2017 Elsevier B.V. All rights reserved.

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

PubMed

Matragkou, Ch; Papachristou, H; Karetsou, Z; Papadopoulos, G; Papamarcaki, T; Vizirianakis, I S; Tsiftsoglou, A S; Choli-Papadopoulou, T

2009-10-09

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.

Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Sundin, George W; Zhao, Youfu

2016-06-01

The type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by an RpoN-HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant-pathogenic bacteria. Complementation of the hrpL mutant with promoter deletion constructs of the hrpL gene and promoter activity analyses using various lengths of the hrpL promoter fused to a promoter-less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between -138 and -125 nucleotides (TGCAA-N4-TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrpL gene among plant enterobacterial pathogens. Results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and electrophoresis mobility shift assay coupled with site-directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E. amylovora was characterized by complementation of the hrpS mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E. amylovora. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy

PubMed Central

Yang, Qiang; Ma, Yanling; Zhao, Yongxue; She, Zhennan; Wang, Long; Li, Jie; Wang, Chunling; Deng, Yihui

2013-01-01

Background Sequential low-dose chemotherapy has received great attention for its unique advantages in attenuating multidrug resistance of tumor cells. Nevertheless, it runs the risk of producing new problems associated with the accelerated blood clearance phenomenon, especially with multiple injections of PEGylated liposomes. Methods Liposomes were labeled with fluorescent phospholipids of 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and epirubicin (EPI). The pharmacokinetics profile and biodistribution of the drug and liposome carrier following multiple injections were determined. Meanwhile, the antitumor effect of sequential low-dose chemotherapy was tested. To clarify this unexpected phenomenon, the production of polyethylene glycol (PEG)-specific immunoglobulin M (IgM), drug release, and residual complement activity experiments were conducted in serum. Results The first or sequential injections of PEGylated liposomes within a certain dose range induced the rapid clearance of subsequently injected PEGylated liposomal EPI. Of note, the clearance of EPI was two- to three-fold faster than the liposome itself, and a large amount of EPI was released from liposomes in the first 30 minutes in a complement-activation, direct-dependent manner. The therapeutic efficacy of liposomal EPI following 10 days of sequential injections in S180 tumor-bearing mice of 0.75 mg EPI/kg body weight was almost completely abolished between the sixth and tenth day of the sequential injections, even although the subsequently injected doses were doubled. The level of PEG-specific IgM in the blood increased rapidly, with a larger amount of complement being activated while the concentration of EPI in blood and tumor tissue was significantly reduced. Conclusion Our investigation implied that the accelerated blood clearance phenomenon and its accompanying rapid leakage and clearance of drug following sequential low-dose injections may reverse the unique pharmacokinetic–toxicity profile of liposomes which deserved our attention. Therefore, a more reasonable treatment regime should be selected to lessen or even eliminate this phenomenon. PMID:23576868

Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

PubMed

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-05-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin

PubMed Central

Broders-Bondon, Florence; Paul-Gilloteaux, Perrine; Gazquez, Elodie; Heysch, Julie; Piel, Matthieu; Mayor, Roberto; Lambris, John D.; Dufour, Sylvie

2016-01-01

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cad-herin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cad-herin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders. PMID:27041467

Comparative immune responses of corals to stressors associated with offshore reef-based tourist platforms

PubMed Central

Lamb, Joleah B; van Oppen, Madeleine J H; Willis, Bette L; Bourne, David G

2015-01-01

Abstract Unravelling the contributions of local anthropogenic and seasonal environmental factors in suppressing the coral immune system is important for prioritizing management actions at reefs exposed to high levels of human activities. Here, we monitor health of the model coral Acropora millepora adjacent to a high-use and an unused reef-based tourist platform, plus a nearby control site without a platform, over 7 months spanning a typical austral summer. Comparisons of temporal patterns in a range of biochemical and genetic immune parameters (Toll-like receptor signalling pathway, lectin–complement system, prophenoloxidase-activating system and green fluorescent protein-like proteins) among healthy, injured and diseased corals revealed that corals exhibit a diverse array of immune responses to environmental and anthropogenic stressors. In healthy corals at the control site, expression of genes involved in the Toll-like receptor signalling pathway (MAPK p38, MEKK1, cFos and ATF4/5) and complement system (C3 and Bf) was modulated by seasonal environmental factors in summer months. Corals at reef platform sites experienced additional stressors over the summer, as evidenced by increased expression of various immune genes, including MAPK p38 and MEKK1. Despite increased expression of immune genes, signs of white syndromes were detected in 31% of study corals near tourist platforms in the warmest summer month. Evidence that colonies developing disease showed reduced expression of genes involved in the complement pathway prior to disease onset suggests that their immune systems may have been compromised. Responses to disease and physical damage primarily involved the melanization cascade and GFP-like proteins, and appeared to be sufficient for recovery when summer heat stress subsided. Overall, seasonal and anthropogenic factors may have interacted synergistically to overwhelm the immune systems of corals near reef platforms, leading to increased disease prevalence in summer at these sites. PMID:27293717

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation.

PubMed

Heuermann, D; Haas, R

1998-03-01

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (catGC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 x 10(-7) and 4.7 x 10(-7) transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H. pylori recipients, with pHel2 showing an efficiency of 2.0 x 10(-5) transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylori recA+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H. pylori demonstrate the general usefulness of this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.

Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1.

PubMed

Dodd, I; Mossakowska, D E; Camilleri, P; Haran, M; Hensley, P; Lawlor, E J; McBay, D L; Pindar, W; Smith, R A

1995-12-01

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris ▿

PubMed Central

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-01-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells. PMID:21421775

Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.)

PubMed Central

Majer, Christine; Xu, Changzheng; Berendzen, Kenneth W.; Hochholdinger, Frank

2012-01-01

Rootless concerning crown and seminal roots (Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize. PMID:22527397

A biophysical investigation on the binding of proflavine with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM studies.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2016-12-01

Interaction of proflavine with hemoglobin (Hgb) was studied employing spectroscopy, calorimetry, and atomic force microscopy. The equilibrium constant was found to be of the order 10 4 M -1 . The quenching of Hgb fluorescence by proflavine was due to the complex formation. Calculation of the molecular distance (r) between the donor (β-Trp37 of Hgb) and acceptor (proflavine) suggested that energy can be efficiently transferred from the β-Trp37 residue at the α1β2 interface of the protein to the dye. Proflavine induced significant secondary structural changes in Hgb. Synchronous fluorescence studies showed that proflavine altered the microenvironment around the tryptophan residues to a greater extent than the tyrosine residues. Circular dichroism spectral studies showed that proflavine caused significant reduction in the α-helical content of Hgb. The esterase activity assay further complemented the circular dichroism data. The Soret band intensity of Hgb decreased upon complexation. Differential scanning calorimetry and circular dichroism melting results revealed that proflavine induced destabilization of Hgb. The binding was driven by both positive entropy and negative enthalpy. Atomic force microscopy studies revealed that the essential morphological features of hemoglobin were retained in the presence of proflavine. Overall, insights on the photophysical aspects and energetics of the binding of proflavine with Hgb are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.

PubMed

Bruno, John Gordon; Carrillo, Maria P; Phillips, Taylor; Hanson, Douglas; Bohmann, Jonathan A

2011-09-01

A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.

In situ investigation of Geobacillus stearothermophilus spore germination and inactivation mechanisms under moderate high pressure.

PubMed

Georget, Erika; Kapoor, Shobhna; Winter, Roland; Reineke, Kai; Song, Youye; Callanan, Michael; Ananta, Edwin; Heinz, Volker; Mathys, Alexander

2014-08-01

Bacterial spores are a major concern for food safety due to their high resistance to conventional preservation hurdles. Innovative hurdles can trigger bacterial spore germination or inactivate them. In this work, Geobacillus stearothermophilus spore high pressure (HP) germination and inactivation mechanisms were investigated by in situ infrared spectroscopy (FT-IR) and fluorometry. G. stearothermophilus spores' inner membrane (IM) was stained with Laurdan fluorescent dye. Time-dependent FT-IR and fluorescence spectra were recorded in situ under pressure at different temperatures. The Laurdan spectrum is affected by the lipid packing and level of hydration, and provided information on the IM state through the Laurdan generalized polarization. Changes in the -CH2 and -CH3 asymmetric stretching bands, characteristic of lipids, and in the amide I' band region, characteristic of proteins' secondary structure elements, enabled evaluation of the impact of HP on endospores lipid and protein structures. These studies were complemented by ex situ analyses (plate counts and microscopy). The methods applied showed high potential to identify germination mechanisms, particularly associated to the IM. Germination up to 3 log10 was achieved at 200 MPa and 55 °C. A molecular-level understanding of these mechanisms is important for the development and validation of multi-hurdle approaches to achieve commercial sterility. Copyright © 2014 Elsevier Ltd. All rights reserved.

Human molecular cytogenetics: From cells to nucleotides

PubMed Central

Riegel, Mariluce

2014-01-01

The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH), a technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods, cytogeneticists have used a range of technologies to study the association between visible chromosome rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful and informative complement to other molecular and genomic approaches. This manuscript does not present a detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the technologies used to identify chromosomal rearrangements and copy number changes, and the applications for cytogenetics in biomedical diagnosis and research are presented and discussed. PMID:24764754

Investigation of Antigen-Antibody Interactions of Sulfonamides with a Monoclonal Antibody in a Fluorescence Polarization Immunoassay Using 3D-QSAR Models

PubMed Central

Wang, Zhanhui; Kai, Zhenpeng; Beier, Ross C.; Shen, Jianzhong; Yang, Xinling

2012-01-01

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA). The affinities of the MAbSMR, expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence polarization immunoassay (FPIA). The results demonstrated that the proposed pharmacophore model containing two hydrogen-bond acceptors, two hydrogen-bond donors and two hydrophobic centers characterized the structural features of the sulfonamides necessary for MAbSMR binding. Removal of two outliers from the initial set of 17 sulfonamide analogs improved the predictability of the models. The 3D-QSAR models of 15 sulfonamides based on CoMFA and CoMSIA resulted in q2 cv values of 0.600 and 0.523, and r2 values of 0.995 and 0.994, respectively, which indicates that both methods have significant predictive capability. Connolly surface analysis, which mainly focused on steric force fields, was performed to complement the results from CoMFA and CoMSIA. This novel study combining FPIA with pharmacophore modeling demonstrates that multidisciplinary research is useful for investigating antigen-antibody interactions and also may provide information required for the design of new haptens. PMID:22754368

Molecular interactions of ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS, a LOB domain protein regulating shoot-borne root initiation in maize (Zea mays L.).

PubMed

Majer, Christine; Xu, Changzheng; Berendzen, Kenneth W; Hochholdinger, Frank

2012-06-05

Rootless concerning crown and seminal roots (Rtcs) encodes a LATERAL ORGAN BOUNDARIES domain (LBD) protein that regulates shoot-borne root initiation in maize (Zea mays L.). GREEN FLUORESCENT PROTEIN (GFP)-fusions revealed RTCS localization in the nucleus while its paralogue RTCS-LIKE (RTCL) was detected in the nucleus and cytoplasm probably owing to an amino acid exchange in a nuclear localization signal. Moreover, enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that RTCS primarily binds to LBD DNA motifs. RTCS binding to an LBD motif in the promoter of the auxin response factor (ARF) ZmArf34 and reciprocally, reciprocal ZmARF34 binding to an auxin responsive element motif in the promoter of Rtcs was shown by electrophoretic mobility shift assay experiments. In addition, comparative qRT-PCR of wild-type versus rtcs coleoptilar nodes suggested RTCS-dependent activation of ZmArf34 expression. Consistently, luciferase reporter assays illustrated the capacity of RTCS, RTCL and ZmARF34 to activate downstream gene expression. Finally, RTCL homo- and RTCS/RTCL hetero-interaction were demonstrated in yeast-two-hybrid and bimolecular fluorescence complementation experiments, suggesting a role of these complexes in downstream gene regulation. In summary, the data provide novel insights into the molecular interactions resulting in crown root initiation in maize.

Cell identification using Raman spectroscopy in combination with optical trapping and microfluidics

NASA Astrophysics Data System (ADS)

Krafft, Christoph; Dochow, Sebastian; Beleites, Claudia; Popp, Jürgen

2014-03-01

Cell identification by Raman spectroscopy has evolved to be an attractive complement to established optical techniques. Raman activated cell sorting (RACS) offers prospects to complement the widely applied fluorescence activated cell sorting. RACS can be realized by combination with optical traps and microfluidic devices. The progress of RACS is reported for a cellular model system that can be found in peripheral blood of tumor patients. Lymphocytes and erythrocytes were extracted from blood samples. Breast carcinoma derived tumor cells (MCF-7, BT-20) and acute myeloid leukemia cells (OCI-AML3) were grown in cell cultures. First, Raman images were collected from dried cells on calcium fluoride slides. Support vector machines (SVM) classified 99.7% of the spectra to the correct cell type. Second, a 785 nm laser was used for optical trapping of single cells in aqueous buffer and for excitation of the Raman spectrum. SVM distinguished 1210 spectra of tumor and normal cells with a sensitivity of >99.7% and a specificity of >99.5%. Third, a microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate fibers of an optical trap and sort single cells after Raman based identification with 514 nm for excitation. Forth, the microfluidic chip was fabricated by quartz which improved cell identification results with 785 nm excitation. Here, partial least squares discriminant analysis gave classification rates of 98%. Finally, a Raman-on-chip approach was developed that integrates fibers for trapping, Raman excitation and signal detection in a single compact unit.

Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice.

PubMed

Zang, Baisheng; Li, Haowen; Li, Wenjun; Deng, Xing Wang; Wang, Xiping

2011-08-01

Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.

Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.

PubMed

Yang, G S; Banks, K G; Bonaguro, R J; Wilson, G; Dreolini, L; de Leeuw, C N; Liu, L; Swanson, D J; Goldowitz, D; Holt, R A; Simpson, E M

2009-03-01

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.

Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification.

PubMed

Scharf, Brian; Clement, Cristina C; Yodmuang, Supansa; Urbanska, Aleksandra M; Suadicani, Sylvia O; Aphkhazava, David; Thi, Mia M; Perino, Giorgio; Hardin, John A; Cobelli, Neil; Vunjak-Novakovic, Gordana; Santambrogio, Laura

2013-07-25

Aging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Suppression of OsKu80 results in defects in developmental growth and increased telomere length in rice (Oryza sativa L.).

PubMed

Byun, Mi Young; Cui, Li Hua; Kim, Woo Taek

2015-12-25

The Ku70-Ku80 heterodimer plays a critical role in the maintenance of genomic stability in humans and yeasts. In this report, we identified and characterized OsKu80 in rice, a model monocot crop. OsKu80 forms a heterodimer with OsKu70 in yeast and plant cells, as demonstrated by yeast two-hybrid, in vivo co-immunoprecipitation, and bimolecular fluorescence complementation assays. RNAi-mediated knock-down T3 transgenic rice plants (Ubi:RNAi-OsKu80) displayed a retarded growth phenotype at the post-germination stage. In addition, the Ubi:RNAi-OsKu80 knock-down progeny exhibited noticeably increased telomere length as compared to wild-type rice. These results are discussed with the idea that OsKu80 plays a role in developmental growth and telomere length regulation in rice plants. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bandyopadhyay, Anindya; Kopperud, Kristin; Anderson, Gavin

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine themore » subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.« less

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yuan; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3; Wu, Keqiang

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants.more » We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.« less

Incunabular Immunological Events in Prion Trafficking

PubMed Central

Michel, Brady; Meyerett-Reid, Crystal; Johnson, Theodore; Ferguson, Adam; Wyckoff, Christy; Pulford, Bruce; Bender, Heather; Avery, Anne; Telling, Glenn; Dow, Steven; Zabel, Mark D.

2012-01-01

While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement. PMID:22679554

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekman, Axel A.; Chen, Jian-Hua; Guo, Jessica

In the context of cell biology, the term mesoscale describes length scales ranging from that of an individual cell, down to the size of the molecular machines. In this spatial regime, small building blocks self-organise to form large, functional structures. A comprehensive set of rules governing mesoscale self-organisation has not been established, making the prediction of many cell behaviours difficult, if not impossible. Our knowledge of mesoscale biology comes from experimental data, in particular, imaging. Here, we explore the application of soft X-ray tomography (SXT) to imaging the mesoscale, and describe the structural insights this technology can generate. We alsomore » discuss how SXT imaging is complemented by the addition of correlative fluorescence data measured from the same cell. This combination of two discrete imaging modalities produces a 3D view of the cell that blends high-resolution structural information with precise molecular localisation data.« less

Arabidopsis mitochondrial voltage-dependent anion channel 3 (AtVDAC3) protein interacts with thioredoxin m2.

PubMed

Zhang, Min; Takano, Tetsuo; Liu, Shenkui; Zhang, Xinxin

2015-05-08

Voltage-dependent anion channels (VDACs) are conserved mitochondrial outer membrane proteins. A yeast two-hybrid screen identified interaction between Arabidopsis VDAC3 and the chloroplast protein thioredoxin m2 (AtTrx m2). This was confirmed via pull-down assay. A bimolecular fluorescence complementation assay located the interaction in mitochondria. AtVDAC3 and AtTrx m2 transcripts were expressed in multiple tissues and up-regulated by abiotic stress. Under NaCl stress, AtVDAC3 overexpression inhibited growth and increased H2O2 accumulation, while AtTrx m2 overexpression conferred resistance to NaCl and reduced H2O2. Results indicate that both AtVDAC3 and AtTrx m2 are involved in ROS signaling and play opposite roles in NaCl stress response. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Pattern Transitions in Bacterial Oscillating System under Nanofluidic Confinement

NASA Astrophysics Data System (ADS)

Shen, Jie-Pan; Chou, Chia-Fu

2011-03-01

Successful binary fission in E. coli relies on remarkable oscillatory behavior of the MinCDE protein system to determine the exact division site. The most favorable models to explain this fascinating spatiotemporal regulation on dynamic MinDE pattern formation in cells are based on reaction-diffusion scenario. Although not fully understood, geometric factors caused by bacterial morphology play a crucial role in MinDE dynamics. In the present study, bacteria were cultured, confined and reshaped in various micro/nanofluidic devices, to mimic either curvature changes of cell peripherals. Fluorescence imaging was utilized to detail the mode transitions in multiple MinDE patterns. The understanding of the physics in multiple pattern formations is further complemented via in silico modeling. The study synergizes the join merits of in vivo, in vitro and in silico approaches, to grasp the insight of stochastic dynamics inherited from the noisy mesoscopic biophysics. We acknowledge support from the Foresight Project, Academia Sinica.

Inhibitor of MYC identified in a Kröhnke pyridine library

PubMed Central

Hart, Jonathan R.; Garner, Amanda L.; Yu, Jing; Ito, Yoshihiro; Sun, Minghao; Ueno, Lynn; Rhee, Jin-Kyu; Baksh, Michael M.; Stefan, Eduard; Hartl, Markus; Bister, Klaus; Vogt, Peter K.; Janda, Kim D.

2014-01-01

In a fluorescence polarization screen for the MYC–MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC–MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. PMID:25114221

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tuskan, Gerald A; Gunter, Lee E; DiFazio, Stephen P

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequencemore » assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.« less

Electronic structure and optical properties of some anthocyanins extracted from grapes

NASA Astrophysics Data System (ADS)

Iosub, Ion; Kajzar, Francois; Makowska-Janusik, Malgorzata; Meghea, Aurelia; Tane, Alexandrina; Rau, Ileana

2012-08-01

The pallet of applications of natural dyes and pigments is continuously extending as a stringent need to meet the challenges arisen from sustainable development, particularly related to replacing synthetic chemicals by eco-friendly renewable raw materials. In this respect, the well known nutritive and therapeutic properties demonstrated by anthocyanin compounds present in selective plant extracts of numerous fruits, vegetables and flowers could be largely complemented by their characteristics as chromophores and fluorophores with high potential in promising applications in bio-imaging for diagnostic and therapy, but also in electronic and photonic devices, solar cells, etc. In this context, this paper proposes an improved protocol for extraction and HPLC detection of anthocyanins from black grapes. Moreover, in order to exploit their potential to be applied as chromatic and fluorescence molecular probes, the electronic properties have been studied by correlating spectral characteristics with trichromatic parameters, combined with quantum chemistry computation.

Genetics Home Reference: Fanconi anemia

MedlinePlus

... D1 Genetic Testing Registry: Fanconi anemia, complementation group D2 Genetic Testing Registry: Fanconi anemia, complementation group E ... ANEMIA, COMPLEMENTATION GROUP D1 FANCONI ANEMIA, COMPLEMENTATION GROUP D2 FANCONI ANEMIA, COMPLEMENTATION GROUP E FANCONI ANEMIA, COMPLEMENTATION ...

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines.

PubMed

Kim, Kyoung Whun; Jeong, Soyoung; Ahn, Ki Bum; Yang, Jae Seung; Yun, Cheol-Heui; Han, Seung Hyun

2017-12-01

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.

Ultrafast proton shuttling in Psammocora cyan fluorescent protein.

PubMed

Kennis, John T M; van Stokkum, Ivo H M; Peterson, Dayna S; Pandit, Anjali; Wachter, Rebekka M

2013-09-26

Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus Psammocora of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump-dump-probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump-dump-probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20-4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly involving a short hydrogen bond. At any time in the reaction, the proton is localized on either the chromophore or Glu-167, which implies that most likely no low-barrier hydrogen bond exists between these molecular groups. This work supports the notion that proton transfer in biological systems, be it in an electronic excited or ground state, can be an intrinsically fast process that occurs on a 100 fs time scale. PsamFP488 represents an attractive model system that poses an ultrafast proton transfer regime in discrete steps. It constitutes a valuable model system in addition to wild type GFP, where proton transfer is relatively slow, and the S65T/H148D GFP mutant, where the effects of low-barrier hydrogen bonds dominate.

The Complement System in Dialysis: A Forgotten Story?

PubMed Central

Poppelaars, Felix; Faria, Bernardo; Gaya da Costa, Mariana; Franssen, Casper F. M.; van Son, Willem J.; Berger, Stefan P.; Daha, Mohamed R.; Seelen, Marc A.

2018-01-01

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD. PMID:29422906

Complement Activation in Inflammatory Skin Diseases

PubMed Central

Giang, Jenny; Seelen, Marc A. J.; van Doorn, Martijn B. A.; Rissmann, Robert; Prens, Errol P.; Damman, Jeffrey

2018-01-01

The complement system is a fundamental part of the innate immune system, playing a crucial role in host defense against various pathogens, such as bacteria, viruses, and fungi. Activation of complement results in production of several molecules mediating chemotaxis, opsonization, and mast cell degranulation, which can contribute to the elimination of pathogenic organisms and inflammation. Furthermore, the complement system also has regulating properties in inflammatory and immune responses. Complement activity in diseases is rather complex and may involve both aberrant expression of complement and genetic deficiencies of complement components or regulators. The skin represents an active immune organ with complex interactions between cellular components and various mediators. Complement involvement has been associated with several skin diseases, such as psoriasis, lupus erythematosus, cutaneous vasculitis, urticaria, and bullous dermatoses. Several triggers including auto-antibodies and micro-organisms can activate complement, while on the other hand complement deficiencies can contribute to impaired immune complex clearance, leading to disease. This review provides an overview of the role of complement in inflammatory skin diseases and discusses complement factors as potential new targets for therapeutic intervention. PMID:29713318

The Semantics of Complementation in English: A Cognitive Semantic Account of Two English Complement Constructions

ERIC Educational Resources Information Center

Smith, Michael B.

2009-01-01

Studies on complementation in English and other languages have traditionally focused on syntactic issues, most notably on the constituent structures of different complement types. As a result, they have neglected the role of meaning in the choice of different complements. This paper investigates the semantics of complementation within the…

Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

PubMed

Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

2016-02-01

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals. Copyright © 2015 John Wiley & Sons, Ltd.

The Complement System and Adverse Pregnancy Outcomes

PubMed Central

Regal, Jean F.; Gilbert, Jeffrey S.; Burwick, Richard M.

2015-01-01

Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the feta allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child. PMID:25802092

Sundanese Complementation

ERIC Educational Resources Information Center

Kurniawan, Eri

2013-01-01

The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,…

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

PubMed

Callén, E; Tischkowitz, M D; Creus, A; Marcos, R; Bueren, J A; Casado, J A; Mathew, C G; Surrallés, J

2004-01-01

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate. Copyright 2003 S. Karger AG, Basel

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

PubMed Central

Bach, Liên; Michaelson, Louise V.; Haslam, Richard; Bellec, Yannick; Gissot, Lionel; Marion, Jessica; Da Costa, Marco; Boutin, Jean-Pierre; Miquel, Martine; Tellier, Frédérique; Domergue, Frederic; Markham, Jonathan E.; Beaudoin, Frederic; Napier, Johnathan A.; Faure, Jean-Denis

2008-01-01

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes. PMID:18799749

The tick-over theory revisited: formation and regulation of the soluble alternative complement C3 convertase (C3(H2O)Bb).

PubMed

Bexborn, Fredrik; Andersson, Per Ola; Chen, Hui; Nilsson, Bo; Ekdahl, Kristina N

2008-04-01

The molecular interactions between the components of the C3 convertase of the alternative pathway (AP) of complement and its regulators, in both surface-bound and fluid-phase form, are still incompletely understood. The fact that the AP convertase is labile makes studies difficult to perform. According to the so called tick-over theory, hydrolyzed C3, called C3(H(2)O), forms the initial convertase in fluid phase together with factor B. In the present study, we have applied western blot analysis and ELISA together with fluorescence resonance energy transfer (FRET) to study the formation of the fluid-phase AP convertases C3(H(2)O)Bb and C3bBb and their regulation by factor H and factor I at specific time points and, with FRET, in real time. In our hands, factor B showed a higher affinity for C3(H(2)O) than for C3b, although in both cases it was readily activated to Bb. However, the convertase activity of C3bBb was approximately twice that of C3(H(2)O)Bb, as monitored by the generation of C3a. But in contrast, the C3(H(2)O)Bb convertase was more resistant to inactivation by factor H and factor I than was the C3bBb convertase. Under conditions that totally inactivated C3bBb, C3(H(2)O)Bb still retained approximately 25% of its initial activity.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

The Tick-Over Theory Revisited: Formation and Regulation of the Soluble Alternative Complement C3 Convertase (C3(H2O)Bb)

PubMed Central

Bexborn, Fredrik; Andersson, Per Ola; Chen, Hui; Nilsson, Bo; Ekdahl, Kristina N.

2009-01-01

The molecular interactions between the components of the C3 convertase of the alternative pathway (AP) of complement and its regulators, in both surface-bound and fluid-phase form, are still incompletely understood. The fact that the AP convertase is labile makes studies difficult to perform. According to the so called tick-over theory, hydrolyzed C3, called C3(H2O), forms the initial convertase in fluid phase together with factor B. In the present study, we have applied western blot analysis and ELISA together with fluorescence resonance energy transfer (FRET) to study the formation of the fluid-phase AP convertases C3(H2O)Bb and C3bBb and their regulation by factor H and factor I at specific time points and, with FRET, in real time. In our hands, factor B showed a higher affinity for C3(H2O) than for C3b, although in both cases it was readily activated to Bb. However, the convertase activity of C3bBb was approximately twice that of C3(H2O)Bb, as monitored by the generation of C3a. But in contrast, the C3(H2O)Bb convertase was more resistant to inactivation by factor H and factor I than was the C3bBb convertase. Under conditions that totally inactivated C3bBb, C3(H2O)Bb still retained approximately 25% of its initial activity. PMID:18096230

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

Silica-coated quantum dots for optical evaluation of perfluorocarbon droplet interactions with cells.

PubMed

Gorelikov, Ivan; Martin, Amanda L; Seo, Minseok; Matsuura, Naomi

2011-12-20

There has been recent interest in developing new, targeted, perfluorocarbon (PFC) droplet-based contrast agents for medical imaging (e.g., magnetic resonance imaging, X-ray/computed tomography, and ultrasound imaging). However, due to the large number of potential PFCs and droplet stabilization strategies available, it is challenging to determine in advance the PFC droplet formulation that will result in the optimal in vivo behavior and imaging performance required for clinical success. We propose that the integration of fluorescent quantum dots (QDs) into new PFC droplet agents can help to rapidly screen new PFC-based candidate agents for biological compatibility early in their development. QD labels can allow the interaction of PFC droplets with single cells to be assessed at high sensitivity and resolution using optical methods in vitro, complementing the deeper depth penetration but lower resolution provided by PFC droplet imaging using in vivo medical imaging systems. In this work, we introduce a simple and robust method to miscibilize silica-coated nanoparticles into hydrophobic and lipophobic PFCs through fluorination of the silica surface via a hydrolysis-condensation reaction with 1H,1H,2H,2H-perfluorodecyltriethoxysilane. Using CdSe/ZnS core/shell QDs, we show that nanoscale, QD-labeled PFC droplets can be easily formed, with similar sizes and surface charges as unlabeled PFC droplets. The QD label can be used to determine the PFC droplet uptake into cells in vitro by fluorescence microscopy and flow cytometry, and can be used to validate the fate of PFC droplets in vivo in small animals via fluorescence microscopy of histological tissue sections. This is demonstrated in macrophage and cancer cells, and in rabbits, respectively. This work reveals the potential of using QD labels for rapid, preclinical, optical assessment of different PFC droplet formulations for their future use in patients. © 2011 American Chemical Society

DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

PubMed Central

Ross, Martin R.; Borman, Earle K.

1963-01-01

Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

PubMed Central

Kaido, Masanori; Abe, Kazutomo; Mine, Akira; Hyodo, Kiwamu; Taniguchi, Takako; Taniguchi, Hisaaki; Mise, Kazuyuki; Okuno, Tetsuro

2014-01-01

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. PMID:25411849

Automating X-ray Fluorescence Analysis for Rapid Astrobiology Surveys.

PubMed

Thompson, David R; Flannery, David T; Lanka, Ravi; Allwood, Abigail C; Bue, Brian D; Clark, Benton C; Elam, W Timothy; Estlin, Tara A; Hodyss, Robert P; Hurowitz, Joel A; Liu, Yang; Wade, Lawrence A

2015-11-01

A new generation of planetary rover instruments, such as PIXL (Planetary Instrument for X-ray Lithochemistry) and SHERLOC (Scanning Habitable Environments with Raman Luminescence for Organics and Chemicals) selected for the Mars 2020 mission rover payload, aim to map mineralogical and elemental composition in situ at microscopic scales. These instruments will produce large spectral cubes with thousands of channels acquired over thousands of spatial locations, a large potential science yield limited mainly by the time required to acquire a measurement after placement. A secondary bottleneck also faces mission planners after downlink; analysts must interpret the complex data products quickly to inform tactical planning for the next command cycle. This study demonstrates operational approaches to overcome these bottlenecks by specialized early-stage science data processing. Onboard, simple real-time systems can perform a basic compositional assessment, recognizing specific features of interest and optimizing sensor integration time to characterize anomalies. On the ground, statistically motivated visualization can make raw uncalibrated data products more interpretable for tactical decision making. Techniques such as manifold dimensionality reduction can help operators comprehend large databases at a glance, identifying trends and anomalies in data. These onboard and ground-side analyses can complement a quantitative interpretation. We evaluate system performance for the case study of PIXL, an X-ray fluorescence spectrometer. Experiments on three representative samples demonstrate improved methods for onboard and ground-side automation and illustrate new astrobiological science capabilities unavailable in previous planetary instruments. Dimensionality reduction-Planetary science-Visualization.

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis

PubMed Central

1994-01-01

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning. PMID:7929583

Laser Induced Fluorescence Studies of NO Kinetics in Short Pulse Air and Air-Fuel Nonequilibrium Discharges

NASA Astrophysics Data System (ADS)

Lempert, Walter; Uddi, Mruthunjaya; Adamovich, Igor

2008-10-01

Laser Induced Fluorescence is used to measure absolute NO concentrations in air, methane-air, and ethylene-air non-equilibrium plasmas, as a function of time after initiation of a single 25 nsec discharge pulse. Peak NO density in air at 60 torr is ˜8.10^12 cm-3 occurring at ˜500 μs after the pulse, with decay time of ˜16.5 msec. Peak NO atom mole fraction in methane-air at φ=0.5 is approximately equal to that in pure air with similar rise and decay rate. In φ = 0.5 ethylene-air, the rise and decay times are also comparable to air and methane--air, but peak NO concentration is a factor of ˜2.5 lower. Spontaneous emission measurements show that N2(C) and NO (A) decay in ˜25ns and ˜2.5μs, respectively. Kinetic modeling calculations incorporating Boltzmann solver for EEDF, and electron impact and full air species kinetics, complemented with the GRI Mech 3.0 hydrocarbon oxidation mechanism, are compared with the experimental data using three different mechanisms. It is concluded that processes involving long lifetime (˜100 μsec) meta-stable states, such as N2 (X,v) and O2(b^1σ), which are formed by quenching of the metastable N2 (A) state by ground state O2, play a dominant role in NO formation.

Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

PubMed

Kim, Mirim; Kim, Min-Jung; Pandey, Shashank; Kim, Jungmook

2016-11-01

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

PubMed

Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

2012-06-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

Imaging bacteria and biofilms on hardware and periprosthetic tissue in orthopedic infections.

PubMed

Nistico, Laura; Hall-Stoodley, Luanne; Stoodley, Paul

2014-01-01

Infection is a major complication of total joint arthroplasty (TJA) surgery, and even though it is now as low as 1 % in some hospitals, the increasing number of primary surgeries translates to tens of thousands of revisions due to prosthetic joint infection (PJI). In many cases the only solution is revision surgery in which the hardware is removed. This process is extremely long and painful for patients and is a considerable financial burden for the health-care system. A significant proportion of the difficulties in diagnosis and treatment of PJI are associated with biofilm formation where bacteria attach to the surface of the prosthesis and periprosthetic tissue and build a 3-D biofilm community encased in an extracellular polymeric slime (EPS) matrix. Bacteria in biofilms have a low metabolic rate which is thought to be a major contributor to their recalcitrance to antibiotic treatment. The diagnosis of biofilm infections is difficult due to the fact that bacteria in biofilms are not readily cultured with standard clinical microbiology techniques. To identify and visualize in situ biofilm bacteria in orthopedic samples, we have developed protocols for the collection of samples in the operating room, for molecular fluorescent staining with 16S rRNA fluorescence in situ hybridization (FISH), and for imaging of samples using confocal laser scanning microscopy (CLSM). Direct imaging is the only method which can definitively identify biofilms on implants and complements both culture and culture-independent diagnostic methods.

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

PubMed Central

Seo, Jang-Kyun; Kwon, Sun-Jung

2012-01-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity. PMID:22438552

A novel method to detect unlabeled inorganic nanoparticles and submicron particles in tissue by sedimentation field-flow fractionation

PubMed Central

Deering, Cassandra E; Tadjiki, Soheyl; Assemi, Shoeleh; Miller, Jan D; Yost, Garold S; Veranth, John M

2008-01-01

A novel methodology to detect unlabeled inorganic nanoparticles was experimentally demonstrated using a mixture of nano-sized (70 nm) and submicron (250 nm) silicon dioxide particles added to mammalian tissue. The size and concentration of environmentally relevant inorganic particles in a tissue sample can be determined by a procedure consisting of matrix digestion, particle recovery by centrifugation, size separation by sedimentation field-flow fractionation (SdFFF), and detection by light scattering. Background Laboratory nanoparticles that have been labeled by fluorescence, radioactivity, or rare elements have provided important information regarding nanoparticle uptake and translocation, but most nanomaterials that are commercially produced for industrial and consumer applications do not contain a specific label. Methods Both nitric acid digestion and enzyme digestion were tested with liver and lung tissue as well as with cultured cells. Tissue processing with a mixture of protease enzymes is preferred because it is applicable to a wide range of particle compositions. Samples were visualized via fluorescence microscopy and transmission electron microscopy to validate the SdFFF results. We describe in detail the tissue preparation procedures and discuss method sensitivity compared to reported levels of nanoparticles in vivo. Conclusion Tissue digestion and SdFFF complement existing techniques by precisely identifying unlabeled metal oxide nanoparticles and unambiguously distinguishing nanoparticles (diameter<100 nm) from both soluble compounds and from larger particles of the same nominal elemental composition. This is an exciting capability that can facilitate epidemiological and toxicological research on natural and manufactured nanomaterials. PMID:19055780

Concentration of atomic hydrogen in a dielectric barrier discharge measured by two-photon absorption fluorescence

NASA Astrophysics Data System (ADS)

Dvořák, P.; Talába, M.; Obrusník, A.; Kratzer, J.; Dědina, J.

2017-08-01

Two-photon absorption laser-induced fluorescence (TALIF) was utilized for measuring the concentration of atomic hydrogen in a volume dielectric barrier discharge (DBD) ignited in mixtures of Ar, H2 and O2 at atmospheric pressure. The method was calibrated by TALIF of krypton diluted in argon at atmospheric pressure, proving that three-body collisions had a negligible effect on quenching of excited krypton atoms. The diagnostic study was complemented with a 3D numerical model of the gas flow and a zero-dimensional model of the chemistry in order to better understand the reaction kinetics and identify the key pathways leading to the production and destruction of atomic hydrogen. It was determined that the density of atomic hydrogen in Ar-H2 mixtures was in the order of 1021 m-3 and decreased when oxygen was added into the gas mixture. Spatially resolved measurements and simulations revealed a sharply bordered region with low atomic hydrogen concentration when oxygen was added to the gas mixture. At substoichiometric oxygen/hydrogen ratios, this H-poor region is confined to an area close to the gas inlet and it is shown that the size of this region is not only influenced by the chemistry but also by the gas flow patterns. Experimentally, it was observed that a decrease in H2 concentration in the feeding Ar-H2 mixture led to an increase in H production in the DBD.

Let’s Tie the Knot: Marriage of Complement and Adaptive Immunity in Pathogen Evasion, for Better or Worse

PubMed Central

Bennett, Kaila M.; Rooijakkers, Suzan H. M.; Gorham, Ronald D.

2017-01-01

The complement system is typically regarded as an effector arm of innate immunity, leading to recognition and killing of microbial invaders in body fluids. Consequently, pathogens have engaged in an arms race, evolving molecules that can interfere with proper complement responses. However, complement is no longer viewed as an isolated system, and links with other immune mechanisms are continually being discovered. Complement forms an important bridge between innate and adaptive immunity. While its roles in innate immunity are well-documented, its function in adaptive immunity is less characterized. Therefore, it is no surprise that the field of pathogenic complement evasion has focused on blockade of innate effector functions, while potential inhibition of adaptive immune responses (via complement) has been overlooked to a certain extent. In this review, we highlight past and recent developments on the involvement of complement in the adaptive immune response. We discuss the mechanisms by which complement aids in lymphocyte stimulation and regulation, as well as in antigen presentation. In addition, we discuss microbial complement evasion strategies, and highlight specific examples in the context of adaptive immune responses. These emerging ties between complement and adaptive immunity provide a catalyst for future discovery in not only the field of adaptive immune evasion but in elucidating new roles of complement. PMID:28197139

Let's Tie the Knot: Marriage of Complement and Adaptive Immunity in Pathogen Evasion, for Better or Worse.

PubMed

Bennett, Kaila M; Rooijakkers, Suzan H M; Gorham, Ronald D

2017-01-01

The complement system is typically regarded as an effector arm of innate immunity, leading to recognition and killing of microbial invaders in body fluids. Consequently, pathogens have engaged in an arms race, evolving molecules that can interfere with proper complement responses. However, complement is no longer viewed as an isolated system, and links with other immune mechanisms are continually being discovered. Complement forms an important bridge between innate and adaptive immunity. While its roles in innate immunity are well-documented, its function in adaptive immunity is less characterized. Therefore, it is no surprise that the field of pathogenic complement evasion has focused on blockade of innate effector functions, while potential inhibition of adaptive immune responses (via complement) has been overlooked to a certain extent. In this review, we highlight past and recent developments on the involvement of complement in the adaptive immune response. We discuss the mechanisms by which complement aids in lymphocyte stimulation and regulation, as well as in antigen presentation. In addition, we discuss microbial complement evasion strategies, and highlight specific examples in the context of adaptive immune responses. These emerging ties between complement and adaptive immunity provide a catalyst for future discovery in not only the field of adaptive immune evasion but in elucidating new roles of complement.

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

PubMed Central

Hecker, Laura A.; Edwards, Albert O.; Ryu, Euijung; Tosakulwong, Nirubol; Baratz, Keith H.; Brown, William L.; Issa, Peter Charbel; Scholl, Hendrik P.; Pollok-Kopp, Beatrix; Schmid-Kubista, Katharina E.; Bailey, Kent R.; Oppermann, Martin

2010-01-01

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues. PMID:19825847

Keeping It All Going-Complement Meets Metabolism.

PubMed

Kolev, Martin; Kemper, Claudia

2017-01-01

The complement system is an evolutionary old and crucial component of innate immunity, which is key to the detection and removal of invading pathogens. It was initially discovered as a liver-derived sentinel system circulating in serum, the lymph, and interstitial fluids that mediate the opsonization and lytic killing of bacteria, fungi, and viruses and the initiation of the general inflammatory responses. Although work performed specifically in the last five decades identified complement also as a critical instructor of adaptive immunity-indicating that complement's function is likely broader than initially anticipated-the dominant opinion among researchers and clinicians was that the key complement functions were in principle defined. However, there is now a growing realization that complement activity goes well beyond "classic" immune functions and that this system is also required for normal (neuronal) development and activity and general cell and tissue integrity and homeostasis. Furthermore, the recent discovery that complement activation is not confined to the extracellular space but occurs within cells led to the surprising understanding that complement is involved in the regulation of basic processes of the cell, particularly those of metabolic nature-mostly via novel crosstalks between complement and intracellular sensor, and effector, pathways that had been overlooked because of their spatial separation. These paradigm shifts in the field led to a renaissance in complement research and provide new platforms to now better understand the molecular pathways underlying the wide-reaching effects of complement functions in immunity and beyond. In this review, we will cover the current knowledge about complement's emerging relationship with the cellular metabolism machinery with a focus on the functional differences between serum-circulating versus intracellularly active complement during normal cell survival and induction of effector functions. We will also discuss how taking a closer look into the evolution of key complement components not only made the functional connection between complement and metabolism rather "predictable" but how it may also give clues for the discovery of additional roles for complement in basic cellular processes.

Complement in Lupus Nephritis: New Perspectives.

PubMed

Bao, Lihua; Cunningham, Patrick N; Quigg, Richard J

2015-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical trials support the use of complement-targeted therapy in the treatment of SLE.

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

PubMed Central

Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

2009-01-01

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

PubMed Central

Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

2016-01-01

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

Blood SC5b-9 complement levels increase at parturition during term and preterm labor.

PubMed

Segura-Cervantes, Enrique; Mancilla-Ramirez, Javier; Zurita, Luis; Paredes, Yuriria; Arredondo, José Luis; Galindo-Sevilla, Norma

2015-06-01

We explored the hypothesis that complement, an innate and adaptive immune effector, is active in the plasma of parturient women and is deposited on fetal membranes collected after delivery. A cross-sectional study was designed to evaluate complement activity at parturition. Pregnant women (n = 97) between 15 and 41 years of age were enrolled in a hospital protocol during the perinatal period to assess both SC5b-9 complement activity in blood and complement deposition on fetal membranes during parturition. Soluble SC5b-9 complement activity in plasma fractions was measured using a standard enzyme-linked immunosorbent assay (ELISA) that included specific anti-complement antibodies. Complement deposition on membranes was analyzed using immuno-dot blots and immunohistochemistry. Soluble SC5b-9 complement complex levels were increased in the plasma of women during term labor (TL; median 3361; range 1726-5670 ng/mL), preterm labor (PL; median 2958; range 1552-7092 ng/mL), and preterm premature rupture of membranes (PPROM; median 2272; range 167-6540 ng/mL) compared with pregnant women who were not in labor (P; median 1384; range 174-4570 ng/mL; P < 0.001, Kruskal-Wallis test). Active complement, as assessed by the C9 neo-antigen in C5b-9 complexes, was deposited on fetal membranes, with no difference between term and preterm delivery. The deposition of active complement on fetal membranes was confirmed by immunohistochemistry. Women who underwent non-labor-indicated Cesarean sections did not exhibit complement deposition. Soluble SC5b-9 complement complex levels increased in the plasma of women during parturition, and complement C5b-9 complexes were deposited on fetal membranes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

PubMed Central

Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

Complement Constructions in English: Fairly Difficult for EFL Language Learners

ERIC Educational Resources Information Center

Fazeli, Fatemeh; Shokrpour, Nasrin

2012-01-01

Complement constructions vary significantly in English and Persian. There are more complementation structures in English than in Persian and a complement structure in Persian might have more than one equivalent in English. Producing complement structures (CSs) in English is very difficult for native speakers of Persian, especially in an EFL…

Complement fixation test to C burnetii

MedlinePlus

... complement fixation test; Coxiella burnetii - complement fixation test; C burnetii - complement fixation test ... a specific foreign substance ( antigen ), in this case, C burnetii . Antibodies defend the body against bacteria, viruses, ...

Using animal models to determine the significance of complement activation in Alzheimer's disease

PubMed Central

Loeffler, David A

2004-01-01

Complement inflammation is a major inflammatory mechanism whose function is to promote the removal of microorganisms and the processing of immune complexes. Numerous studies have provided evidence for an increase in this process in areas of pathology in the Alzheimer's disease (AD) brain. Because complement activation proteins have been demonstrated in vitro to exert both neuroprotective and neurotoxic effects, the significance of this process in the development and progression of AD is unclear. Studies in animal models of AD, in which brain complement activation can be experimentally altered, should be of value for clarifying this issue. However, surprisingly little is known about complement activation in the transgenic animal models that are popular for studying this disorder. An optimal animal model for studying the significance of complement activation on Alzheimer's – related neuropathology should have complete complement activation associated with senile plaques, neurofibrillary tangles (if present), and dystrophic neurites. Other desirable features include both classical and alternative pathway activation, increased neuronal synthesis of native complement proteins, and evidence for an increase in complement activation prior to the development of extensive pathology. In order to determine the suitability of different animal models for studying the role of complement activation in AD, the extent of complement activation and its association with neuropathology in these models must be understood. PMID:15479474

The complex nature of calcium cation interactions with phospholipid bilayers

PubMed Central

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-01-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association. PMID:27905555

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

2014-01-01

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

FUSE Cycle 3 Program CO22: Chromospheric Activity in Population II Giants

NASA Technical Reports Server (NTRS)

Harper, Graham M.

2004-01-01

One of the mysteries of Population II giants is that they still show chromospheric emission despite their great age. The global dynamo which was active during their main-sequence lifetimes is expected to become extremely weak through magnetic rotational braking. The nature of the observed emission is not understood; although acoustic shock waves might provide the heating, acoustic waves are not predicted to drive the observed mass loss - which in turn requires the dissipation of magneto-hydrodynamic waves. This program was designed to search for the faint stellar H Ly(beta) emission wings and the fluorescent Fe II and H2 emission from one of the brightest, metal poor, Population II stars. These FUSE diagnostics, when combined with existing UV and optical spectra, help determine the major radiative cooling channels for the chromosphere. This observation was to complement that previously planned for the mildly metal deficient giant alpha Boo (K2 III). However, a Boo has yet to be observed with FUSE.

Chromospheric Activity in Population II Giants

NASA Technical Reports Server (NTRS)

Harper, Graham M.

2004-01-01

One of the mysteries of Population II giants is that they still show chromospheric emission despite their great age. The global dynamo which was active during their main-sequence lifetimes is expected to become extremely weak through magnetic rotational braking. The nature of the observed emission is not understood; although acoustic shock waves might provide the heating, acoustic waves are not predicted to drive the observed mass loss - which in turn requires the dissipation of magneto-hydrodynamic waves. This program was designed to search for the faint stellar H Ly beta emission wings and the fluorescent Fe II and H2 emission from one of the brightest, metal poor, Population II stars. These FUSE diagnostics, when combined with existing UV and optical spectra, help determine the major radiative cooling channels for the chromosphere. This observation was to complement that previously planned for the mildly metal deficient giant alpha Boo (K2 III). However, alpha Boo has yet to be observed with FUSE.

Single-cell metabolomics: analytical and biological perspectives.

PubMed

Zenobi, R

2013-12-06

There is currently much interest in broad molecular profiling of single cells; a cell's metabolome-its full complement of small-molecule metabolites-is a direct indicator of phenotypic diversity of single cells and a nearly immediate readout of how cells react to environmental influences. However, the metabolome is very difficult to measure at the single-cell level because of rapid metabolic dynamics, the structural diversity of the molecules, and the inability to amplify or tag small-molecule metabolites. Measurement techniques including mass spectrometry, capillary electrophoresis, and, to a lesser extent, optical spectroscopy and fluorescence detection have led to impressive advances in single-cell metabolomics. Even though none of these methodologies can currently measure the metabolome of a single cell completely, rapidly, and nondestructively, progress has been sufficient such that the field is witnessing a shift from feasibility studies to investigations that yield new biological insight. Particularly interesting fields of application are cancer biology, stem cell research, and monitoring of xenobiotics and drugs in tissue sections at the single-cell level.

Sounding rocket flight report, MUMP 9 and MUMP 10

NASA Technical Reports Server (NTRS)

Grassl, H. J.

1971-01-01

The results of the launching of two-Marshall-University of Michigan Probes (MUMP 9 and MUMP 10), Nike-Tomahawk sounding rocket payloads, are summarized. The MUMP is similar to the thermosphere probe, an ejectable instrument package for studying the variability of the earth's atmospheric parameters. The MUMP 9 payload included an omegatron mass analyzer, a molecular fluorescence densitometer, a mini-tilty filter, and a lunar position sensor. This complement of instruments permitted the determination of the molecular nitrogen density and temperature in the altitude range from approximately 143 to 297 km over Wallops Island, Virginia, during January 1971. The MUMP 10 payload included an omegatron mass analyzer, an electron temperature probe, a cryogenic densitometer, and a solar position sensor. These instruments permitted the determination of the molecular nitrogen density and temperature and the charged particle density and temperature in the altitude range from approximately 145 to 290 km over Wallops Island during the afternoon preceding the MUMP 9 launch.

Visualization of Pulmonary Clearance Mechanisms via Noninvasive Optical Imaging Validated by Near-Infrared Flow Cytometry

PubMed Central

Zhou, Haiying; Gunsten, Sean P.; Zhegalova, Natalia G.; Bloch, Sharon; Achilefu, Samuel; Holley, J. Christopher; Schweppe, Daniel; Akers, Walter; Brody, Steven L.; Eades, William; Berezin, Mikhail Y.

2016-01-01

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as four-hours post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies. PMID:25808737

Prenatal identification of i(Yp) by molecular cytogenetic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, B.T.; Peng, W.; Williams, J. III

1994-09-01

An isochromosome derived from the short arm of the Y chromosome, i(Yp), is a rare marker chromosome. Its de novo presence prenatally represents a diagnostic dilemna since its impact on fetal development is difficult to predict. We present a case of 46,X,+i(Yp) de novo detected in an amniotic fluid specimen received for karyotype analysis. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes including a Y-centromere probe, a Y whole chromosome painting probe, and a lambda HAM2 probe containing 19 kb of AMG-Y sequence, located to Yp11.2, have identified the marker chromosome as i(Yp). Themore » breakpoint on this marker chromosome is tentatively assigned to Yq11.1 which is close to the centromere. The present report illustrates the importance of FISH techniques as a complement to cytogenetic methods for accurate identification of chromosome rearrangements in prenatal diagnosis and genetic counseling.« less

Mesoscale imaging with cryo-light and X-rays: Larger than molecular machines, smaller than a cell: Mesoscale imaging with cryo-light and X-rays

DOE PAGES

Ekman, Axel A.; Chen, Jian-Hua; Guo, Jessica; ...

2016-11-14

In the context of cell biology, the term mesoscale describes length scales ranging from that of an individual cell, down to the size of the molecular machines. In this spatial regime, small building blocks self-organise to form large, functional structures. A comprehensive set of rules governing mesoscale self-organisation has not been established, making the prediction of many cell behaviours difficult, if not impossible. Our knowledge of mesoscale biology comes from experimental data, in particular, imaging. Here, we explore the application of soft X-ray tomography (SXT) to imaging the mesoscale, and describe the structural insights this technology can generate. We alsomore » discuss how SXT imaging is complemented by the addition of correlative fluorescence data measured from the same cell. This combination of two discrete imaging modalities produces a 3D view of the cell that blends high-resolution structural information with precise molecular localisation data.« less

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Competition for space during bacterial colonization of a surface.

PubMed

Lloyd, Diarmuid P; Allen, Rosalind J

2015-09-06

Competition for space is ubiquitous in the ecology of both microorganisms and macro-organisms. We introduce a bacterial model system in which the factors influencing competition for space during colonization of an initially empty habitat can be tracked directly. Using fluorescence microscopy, we follow the fate of individual Escherichia coli bacterial cell lineages as they undergo expansion competition (the race to be the first to colonize a previously empty territory), and as they later compete at boundaries between clonal territories. Our experiments are complemented by computer simulations of a lattice-based model. We find that both expansion competition, manifested as differences in individual cell lag times, and boundary competition, manifested as effects of neighbour cell geometry, can play a role in colonization success, particularly when lineages expand exponentially. This work provides a baseline for investigating how ecological interactions affect colonization of space by bacterial populations, and highlights the potential of bacterial model systems for the testing and development of ecological theory. © 2015 The Authors.

Mechanism of Fusion Triggering by Human Parainfluenza Virus Type III

PubMed Central

Porotto, Matteo; Palmer, Samantha G.; Palermo, Laura M.; Moscona, Anne

2012-01-01

Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection. PMID:22110138

Inheritance of a Balanced t(12;20)(q24.33;p12.2) and Unbalanced der(13)t(7;13)(p21.3;q33.2) from a Maternally Derived Double Balanced Translocation Carrier.

PubMed

Peterson, Jess F; Geddes, Gabrielle C; Basel, Donald G; Schippman, Dana; Grignon, John W; vanTuinen, Peter; Kappes, Ulrike P

2018-03-01

We report a 4-month-old male proband with a history of prominent forehead, hypertelorism, ear abnormalities, micrognathia, hypospadias, and multiple cardiac abnormalities. Initial microarray analysis detected a concurrent 7p21.3-p22.3 duplication and 13q33.2-q34 deletion indicating an unbalanced rearrangement. However, subsequent conventional cytogenetic studies only revealed what appeared to be a balanced t(12;20)(q24.33;p12.2). Fluorescence in situ hybridization (FISH) using chromosome-specific subtelomere probes confirmed the presence of an unbalanced der(13)t(7;13)(p21.3;q33.2) and balanced t(12;20)(q24.33;p12.2), both of maternal origin. In addition to our unique clinical findings, this case highlights the benefits and limitations of both conventional cytogenetic studies and microarray analysis and how FISH complements each methodology.

Competition for space during bacterial colonization of a surface

PubMed Central

Lloyd, Diarmuid P.; Allen, Rosalind J.

2015-01-01

Competition for space is ubiquitous in the ecology of both microorganisms and macro-organisms. We introduce a bacterial model system in which the factors influencing competition for space during colonization of an initially empty habitat can be tracked directly. Using fluorescence microscopy, we follow the fate of individual Escherichia coli bacterial cell lineages as they undergo expansion competition (the race to be the first to colonize a previously empty territory), and as they later compete at boundaries between clonal territories. Our experiments are complemented by computer simulations of a lattice-based model. We find that both expansion competition, manifested as differences in individual cell lag times, and boundary competition, manifested as effects of neighbour cell geometry, can play a role in colonization success, particularly when lineages expand exponentially. This work provides a baseline for investigating how ecological interactions affect colonization of space by bacterial populations, and highlights the potential of bacterial model systems for the testing and development of ecological theory. PMID:26333814

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624.

PubMed

Yamagishi, Kenji; Kimura, Toshiyuki; Oita, Sigeru; Sugiura, Tatsuki; Hirai, Hirofumi

2007-10-01

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 x 10(7) protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.

The complex nature of calcium cation interactions with phospholipid bilayers

NASA Astrophysics Data System (ADS)

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-12-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

MicroXRF tomographic visualization of zinc and iron in the zebrafish embryo at the onset of the hatching period

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan

2016-01-01

Transition metals such as zinc, copper, and iron play key roles in cellular proliferation, cell differentiation, growth, and development. Over the past decade, advances in synchrotron X-ray fluorescence instrumentation presented new opportunities for the three-dimensional mapping of trace metal distributions within intact specimens. Taking advantage of microXRF tomography, we visualized the 3D distribution of zinc and iron in a zebrafish embryo at the onset of the hatching period. The reconstructed volumetric data revealed distinct differences in the elemental distributions, with zinc predominantly localized to the yolk and yolk extension, and iron to various regions of the brain as well asmore » the myotome extending along the dorsal side of the embryo. The data set complements an earlier tomographic study of an embryo at the pharyngula stage (24 hpf), thus offering new insights into the trace metal distribution at key stages of embryonic development.« less

Dynamic contrast optical coherence tomography images transit time and quantifies microvascular plasma volume and flow in the retina and choriocapillaris

PubMed Central

Merkle, Conrad W.; Leahy, Conor; Srinivasan, Vivek J.

2016-01-01

Despite the prevalence of optical imaging techniques to measure hemodynamics in large retinal vessels, quantitative measurements of retinal capillary and choroidal hemodynamics have traditionally been challenging. Here, a new imaging technique called dynamic contrast optical coherence tomography (DyC-OCT) is applied in the rat eye to study microvascular blood flow in individual retinal and choroidal layers in vivo. DyC-OCT is based on imaging the transit of an intravascular tracer dynamically as it passes through the field-of-view. Hemodynamic parameters can be determined through quantitative analysis of tracer kinetics. In addition to enabling depth-resolved transit time, volume, and flow measurements, the injected tracer also enhances OCT angiograms and enables clear visualization of the choriocapillaris, particularly when combined with a post-processing method for vessel enhancement. DyC-OCT complements conventional OCT angiography through quantification of tracer dynamics, similar to fluorescence angiography, but with the important added benefit of laminar resolution. PMID:27867732

Dynamic contrast optical coherence tomography images transit time and quantifies microvascular plasma volume and flow in the retina and choriocapillaris.

PubMed

Merkle, Conrad W; Leahy, Conor; Srinivasan, Vivek J

2016-10-01

Despite the prevalence of optical imaging techniques to measure hemodynamics in large retinal vessels, quantitative measurements of retinal capillary and choroidal hemodynamics have traditionally been challenging. Here, a new imaging technique called dynamic contrast optical coherence tomography (DyC-OCT) is applied in the rat eye to study microvascular blood flow in individual retinal and choroidal layers in vivo . DyC-OCT is based on imaging the transit of an intravascular tracer dynamically as it passes through the field-of-view. Hemodynamic parameters can be determined through quantitative analysis of tracer kinetics. In addition to enabling depth-resolved transit time, volume, and flow measurements, the injected tracer also enhances OCT angiograms and enables clear visualization of the choriocapillaris, particularly when combined with a post-processing method for vessel enhancement. DyC-OCT complements conventional OCT angiography through quantification of tracer dynamics, similar to fluorescence angiography, but with the important added benefit of laminar resolution.

Effect of Pressure on Valence and Structural Properties of YbFe 2 Ge 2 Heavy Fermion Compound—A Combined Inelastic X-ray Spectroscopy, X-ray Diffraction, and Theoretical Investigation

DOE PAGES

Kumar, Ravhi S.; Svane, Axel; Vaitheeswaran, Ganapathy; ...

2015-10-19

We measured the crystal structure and the Yb valence of the YbFe 2Ge 2 heavy fermion compound at room temperature and under high pressures using high-pressure powder X-ray diffraction and X-ray absorption spectroscopy via both partial fluorescence yield and resonant inelastic X-ray emission techniques. Moreover, the measurements are complemented by first-principles density functional theoretical calculations using the self-interaction corrected local spin density approximation investigating in particular the magnetic structure and the Yb valence. Finally, while the ThCr 2Si 2-type tetragonal (I4/mmm) structure is stable up to 53 GPa, the X-ray emission results show an increase of the Yb valence frommore » v = 2.72(2) at ambient pressure to v = 2.93(3) at ~9 GPa, where at low temperature a pressure-induced quantum critical state was reported.« less

Novel method for high-throughput colony PCR screening in nanoliter-reactors

PubMed Central

Walser, Marcel; Pellaux, Rene; Meyer, Andreas; Bechtold, Matthias; Vanderschuren, Herve; Reinhardt, Richard; Magyar, Joseph; Panke, Sven; Held, Martin

2009-01-01

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. PMID:19282448

Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein.

PubMed

Aparicio, Frederic; Pallás, Vicente; Sánchez-Navarro, Jesús

2010-07-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

PubMed

Biró, E; van den Goor, J M; de Mol, B A; Schaap, M C; Ko, L-Y; Sturk, A; Hack, C E; Nieuwland, R

2011-01-01

To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Complement component 4

MedlinePlus

... of a certain protein. This protein is part of the complement system. The complement system is a group of proteins ... system and play a role in the development of inflammation. The complement system protects the body from infections, dead cells and ...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

Landscape complementation revealed through bipartite networks: An example with the Florida manatee

USGS Publications Warehouse

Haase, Catherine G.; Fletcher, Robert J.; Slone, Daniel H.; Reid, James P.; Butler, Susan M.

2017-01-01

Landscape complementation is an important predictor of selection and thus classic complementation measures are not sufficient in describing the process. Formalization of complementation with bipartite network can therefor reveal effects potentially missed with conventional measures.

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

Complement component 3 (C3)

MedlinePlus

... of a certain protein. This protein is part of the complement system. The complement system is a group of proteins ... system and play a role in the development of inflammation. The complement system protects the body from infections, dead cells and ...

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials.

PubMed

Quach, Quang Huy; Kah, James Chen Yong

2017-04-01

The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

PubMed

Jongerius, Ilse; Schuijt, Tim J; Mooi, Frits R; Pinelli, Elena

2015-04-01

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona.

PubMed

Coty, Jean-Baptiste; Eleamen Oliveira, Elquio; Vauthier, Christine

2017-11-05

The understanding of complement activation by nanomaterials is a key to a rational design of safe and efficient nanomedicines. This work proposed a systematic study investigating how molecular design of nanoparticle coronas made of dextran impacts on mechanisms that trigger complement activation. The nanoparticles used for this work consisted of dextran-coated poly(isobutylcyanoacrylate) (PIBCA) nanoparticles have already been thoroughly characterized. Their different capacity to trigger complement activation established on the cleavage of the protein C3 was also already described making these nanoparticles good models to investigate the relation between the molecular feature of their corona and the mechanism by which they triggered complement activation. Results of this new study show that complement activation pathways can be selected by distinct architectures formed by dextran chains composing the nanoparticle corona. Assumptions that explain the relation between complement activation mechanisms triggered by the nanoparticles and the nanoparticle corona molecular feature were proposed. These results are of interest to better understand how the design of dextran-coated nanomaterials will impact interactions with the complement system. It can open perspectives with regard to the selection of a preferential complement activation pathway or prevent the nanoparticles to activate the complement system, based on a rational choice of the corona configuration. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel roles of complement in renal diseases and their therapeutic consequences.

PubMed

Wada, Takehiko; Nangaku, Masaomi

2013-09-01

The complement system functions as a part of the innate immune system. Inappropriate activation of the complement pathways has a deleterious effect on kidneys. Recent advances in complement research have provided new insights into the pathogenesis of glomerular and tubulointerstitial injury associated with complement activation. A new disease entity termed 'C3 glomerulopathy' has recently been proposed and is characterized by isolated C3 deposition in glomeruli without positive staining for immunoglobulins. Genetic and functional studies have demonstrated that several different mutations and disease variants, as well as the generation of autoantibodies, are potentially associated with its pathogenesis. The data from comprehensive analyses suggest that complement dysregulation can also be associated with hemolytic uremic syndrome and more common glomerular diseases, such as IgA nephropathy and diabetic kidney disease. In addition, animal studies utilizing genetically modified mice have begun to elucidate the molecular pathomechanisms associated with the complement system. From a diagnostic point of view, a noninvasive, MRI-based method for detecting C3 has recently been developed to serve as a novel tool for diagnosing complement-mediated kidney diseases. While novel therapeutic tools related to complement regulation are emerging, studies evaluating the precise roles of the complement system in kidney diseases will still be useful for developing new therapeutic approaches.

THE PATHOPHYSIOLOGY OF GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION AND THE COMPLEMENT PATHWAY AS A THERAPEUTIC TARGET

PubMed Central

Schmidt-Erfurth, Ursula; van Lookeren Campagne, Menno; Henry, Erin C.; Brittain, Christopher

2017-01-01

Purpose: Geographic atrophy (GA) is an advanced, vision-threatening form of age-related macular degeneration (AMD) affecting approximately five million individuals worldwide. To date, there are no approved therapeutics for GA treatment; however, several are in clinical trials. This review focuses on the pathophysiology of GA, particularly the role of complement cascade dysregulation and emerging therapies targeting the complement cascade. Methods: Primary literature search on PubMed for GA, complement cascade in age-related macular degeneration. ClinicalTrials.gov was searched for natural history studies in GA and clinical trials of drugs targeting the complement cascade for GA. Results: Cumulative damage to the retina by aging, environmental stress, and other factors triggers inflammation via multiple pathways, including the complement cascade. When regulatory components in these pathways are compromised, as with several GA-linked genetic risk factors in the complement cascade, chronic inflammation can ultimately lead to the retinal cell death characteristic of GA. Complement inhibition has been identified as a key candidate for therapeutic intervention, and drugs targeting the complement pathway are currently in clinical trials. Conclusion: The complement cascade is a strategic target for GA therapy. Further research, including on natural history and genetics, is crucial to expand the understanding of GA pathophysiology and identify effective therapeutic targets. PMID:27902638

Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid.

PubMed

Colombo, Graziano; Clerici, Marco; Altomare, Alessandra; Rusconi, Francesco; Giustarini, Daniela; Portinaro, Nicola; Garavaglia, Maria Lisa; Rossi, Ranieri; Dalle-Donne, Isabella; Milzani, Aldo

2017-01-30

In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved. In this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a rice metal tolerance protein OsMTP11 as a manganese transporter

PubMed Central

Zhang, Mei; Liu, Baoxiu

2017-01-01

Metal tolerance proteins (MTPs) are a gene family of cation efflux transporters that occur widely in plants and might serve an essential role in metal homeostasis and tolerance. Our research describes the identification, characterization, and localization of OsMTP11, a member of the MTP family from rice. OsMTP11 was expressed constitutively and universally in different tissues in rice plant. Heterologous expression in yeast showed that OsMTP11 complemented the hypersensitivity of mutant strains to Mn, and also complemented yeast mutants to other metals, including Co and Ni. Real time RT-PCR analysis demonstrated OsMTP11 expression was substantially enhanced following 4 h under Cd, Zn, Ni, and Mn treatments, suggesting possible roles of OsMTP11 involvement in heavy metal stress responses. Promoter analysis by transgenic assays with GUS as a reporter gene and mRNA in situ hybridization experiments showed that OsMTP11 was expressed specifically in conducting tissues in rice. DNA methylation assays of genomic DNA in rice treated with Cd, Zn, Ni, and Mn revealed that decreased DNA methylation levels were present in the OsMTP11 promoter region, which was consistent with OsMTP11 induced-expression patterns resulting from heavy metal stress. This result suggested that DNA methylation is one of major factors regulating expression of OsMTP11 through epigenetic mechanisms. OsMTP11 fused to green fluorescent protein (GFP) localized to the entire onion epidermal cell cytoplasm, while vacuolar membrane exhibited increased GFP signals, consistent with an OsMTP11 function in cation sequestration. Our results indicated that OsMTP11 might play vital roles in Mn and other heavy metal transportation in rice. PMID:28394944

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1

PubMed Central

Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

2017-01-01

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated. PMID:27451975

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

PubMed

Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

2017-02-01

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

Function of glutathione peroxidases in legume root nodules.

PubMed

Matamoros, Manuel A; Saiz, Ana; Peñuelas, Maria; Bustos-Sanmamed, Pilar; Mulet, Jose M; Barja, Maria V; Rouhier, Nicolas; Moore, Marten; James, Euan K; Dietz, Karl-Josef; Becana, Manuel

2015-05-01

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane.

PubMed

Zhang, Ben; Karnik, Rucha; Wang, Yizhou; Wallmeroth, Niklas; Blatt, Michael R; Grefen, Christopher

2015-06-01

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K(+) channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K(+) channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K(+) channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a "hand-off" in channel control between the two SNARE proteins that is woven together with vesicle fusion. © 2015 American Society of Plant Biologists. All rights reserved.

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin

2010-02-12

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold,more » in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.« less

Dimerization of the bacterial effector protein AvrBs3 in the plant cell cytoplasm prior to nuclear import.

PubMed

Gürlebeck, Doreen; Szurek, Boris; Bonas, Ulla

2005-04-01

The effector protein AvrBs3 from the bacterial phytopathogen Xanthomonas campestris pv. vesicatoria is translocated into the plant cell where it specifically induces hypertrophy symptoms or the hypersensitive reaction. Activity of AvrBs3 depends on nuclear localization signals (NLSs) and an acidic activation domain, suggesting a role in regulation of plant transcription. Here, we show that AvrBs3 dimerizes in the plant cell prior to its nuclear import. AvrBs3 deletion derivatives were tested in the yeast two-hybrid system revealing that the repeat region, which confers specific recognition in resistant plants and is crucial for virulence function, is also essential for the self-interaction. GST pull-down assays showed that the AvrBs3-AvrBs3 interaction occurs independent of plant proteins. Coexpression of two different inactive mutant AvrBs3 derivatives in Bs3-resistant pepper plants resulted in 'trans-complementation', i.e., the induction of a hypersensitive reaction. This clearly indicates that AvrBs3-dimerization occurs in planta. Interestingly, 'trans-complementation' was not observed in susceptible plants suggesting that wild-type homodimers are needed for the AvrBs3 virulence function in plants. Furthermore, a green fluorescent protein (GFP) fusion of AvrBs3 deleted in the NLSs (AvrBs3DeltaNLS-GFP), normally localized in the cytoplasm, was imported into the nucleus upon coexpression with wild-type AvrBs3 in Nicotiana benthamiana. Thus, AvrBs3 dimerization takes place in the cytoplasm of the plant cell prior to nuclear import. Given the fact that dimerization is a common feature of transcriptional regulators, our data are consistent with the idea that AvrBs3 manipulates expression of plant genes involved in the establishment of compatible and incompatible interactions.

Granulocyte and monocyte CD11b expression during plasma separation is dependent on complement factor 5 (C5) - an ex vivo study with blood from a C5-deficient individual.

PubMed

Hardersen, Randolf; Enebakk, Terje; Christiansen, Dorte; Bergseth, Grethe; Brekke, Ole-Lars; Mollnes, Tom Eirik; Lappegård, Knut Tore; Hovland, Anders

2018-04-01

The aim of the study was to investigate the role of complement factor 5 (C5) in reactions elicited by plasma separation using blood from a C5-deficient (C5D) individual, comparing it to C5-deficient blood reconstituted with C5 (C5DR) and blood from healthy donors. Blood was circulated through an ex vivo plasma separation model. Leukocyte CD11b expression and leukocyte-platelet conjugates were measured by flow cytometry during a 30-min period. Other markers were assessed during a 240-min period. Granulocyte and monocyte CD11b expression did not increase in C5D blood during plasma separation. In C5DR samples granulocytes CD11b expression, measured by mean fluorescence intensity (MFI), increased from 10481 ± 6022 (SD) to 62703 ± 4936, and monocytes CD11b expression changed from 13837 ± 7047 to 40063 ± 713. Granulocyte-platelet conjugates showed a 2.5-fold increase in the C5DR sample compared to the C5D sample. Monocyte-platelet conjugates increased independently of C5. In the C5D samples, platelet count decreased from 210 × 10 9 /L (201-219) (median and range) to 51 × 10 9 /L (50-51), and C3bc increased from 14 CAU/mL (21-7) to 198 CAU/mL (127-269), whereas TCC formation was blocked during plasma separation. In conclusion, up-regulation of granulocyte and monocyte CD11b during plasma separation was C5-dependent. The results also indicate C5 dependency in granulocyte-platelet conjugates formation. © 2018 APMIS. Published by John Wiley & Sons Ltd.

On the Functional Overlap between Complement and Anti-Microbial Peptides.

PubMed

Zimmer, Jana; Hobkirk, James; Mohamed, Fatima; Browning, Michael J; Stover, Cordula M

2014-01-01

Intriguingly, activated complement and anti-microbial peptides share certain functionalities; lytic, phagocytic, and chemo-attractant activities and each may, in addition, exert cell instructive roles. Each has been shown to have distinct LPS detoxifying activity and may play a role in the development of endotoxin tolerance. In search of the origin of complement, a functional homolog of complement C3 involved in opsonization has been identified in horseshoe crabs. Horseshoe crabs possess anti-microbial peptides able to bind to acyl chains or phosphate groups/saccharides of endotoxin, LPS. Complement activity as a whole is detectable in marine invertebrates. These are also a source of anti-microbial peptides with potential pharmaceutical applicability. Investigating the locality for the production of complement pathway proteins and their role in modulating cellular immune responses are emerging fields. The significance of local synthesis of complement components is becoming clearer from in vivo studies of parenchymatous disease involving specifically generated, complement-deficient mouse lines. Complement C3 is a central component of complement activation. Its provision by cells of the myeloid lineage varies. Their effector functions in turn are increased in the presence of anti-microbial peptides. This may point to a potentiating range of activities, which should serve the maintenance of health but may also cause disease. Because of the therapeutic implications, this review will consider closely studies dealing with complement activation and anti-microbial peptide activity in acute inflammation (e.g., dialysis-related peritonitis, appendicitis, and ischemia).

Complement System Part II: Role in Immunity

PubMed Central

Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

2015-01-01

The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5240 Complement components immunological test system. (a) Identification. A complement components... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement components immunological test system...

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.

PubMed

Kotimaa, Juha; Klar-Mohammad, Ngaisah; Gueler, Faikah; Schilders, Geurt; Jansen, Aswin; Rutjes, Helma; Daha, Mohamed R; van Kooten, Cees

2016-08-01

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

New Milestones Ahead in Complement-Targeted Therapy

PubMed Central

Ricklin, Daniel; Lambris, John D.

2017-01-01

The complement system is a powerful effector arm of innate immunity that typically confers protection from microbial intruders and accumulating debris. In many clinical situations, however, the defensive functions of complement can turn against host cells and induce or exacerbate immune, inflammatory, and degenerative conditions. Although the value of inhibiting complement in a therapeutic context has long been recognized, bringing complement-targeted drugs into clinical use has proved challenging. This important milestone was finally reached a decade ago, yet the clinical availability of complement inhibitors has remained limited. Still, the positive long-term experience with complement drugs and their proven effectiveness in various diseases has reinvigorated interest and confidence in this approach. Indeed, a broad variety of clinical candidates that act at almost any level of the complement activation cascade are currently in clinical development, with several of them being evaluated in phase 2 and phase 3 trials. With antibody-related drugs dominating the panel of clinical candidates, the emergence of novel small-molecule, peptide, protein, and oligonucleotide-based inhibitors offers new options for drug targeting and administration. Whereas all the currently approved and many of the proposed indications for complement-targeted inhibitors belong to the rare disease spectrum, these drugs are increasingly being evaluated for more prevalent conditions. Fortunately, the growing experience from preclinical and clinical use of therapeutic complement inhibitors has enabled a more evidence-based assessment of suitable targets and rewarding indications as well as related technical and safety considerations. This review highlights recent concepts and developments in complement-targeted drug discovery, provides an overview of current and emerging treatment options, and discusses the new milestones ahead on the way to the next generation of clinically available complement therapeutics. PMID:27321574

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements1[OPEN

PubMed Central

Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; Ries, Amber; Böhmer, Maik; You, Lei; Xiao, Chuanlei; Engineer, Cawas B.; Boron, Walter F.; Schroeder, Julian I.

2015-01-01

Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3− concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3− concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified. PMID:26243620

Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein.

PubMed

Zosel, Franziska; Haenni, Dominik; Soranno, Andrea; Nettels, Daniel; Schuler, Benjamin

2017-10-21

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. To complement the information from FRET, we combine it with photoinduced electron transfer (PET) quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond time scale can be obtained reliably from a single set of measurements by the analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A more detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic molecular dynamics simulation of integrase. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of the dynamics and detailed mechanisms in unfolded and intrinsically disordered proteins.

Efficient formation of heterokaryotic sclerotia in the filamentous fungus Aspergillus oryzae.

PubMed

Wada, Ryuta; Jin, Feng Jie; Koyama, Yasuji; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2014-01-01

Heterokaryon formation by hyphal fusion occurs during a sexual/parasexual cycle in filamentous fungi, and therefore, it is biotechnologically important for crossbreeding. In the industrial filamentous fungus Aspergillus oryzae, a parasexual cycle has been reported, and it was recently suggested that sexual reproduction should be possible. However, as A. oryzae enters into hyphal fusion with a much lower frequency than Neurospora crassa, the process of heterokaryon formation has not been extensively characterized in A. oryzae. Here, we developed a detection system for heterokaryon formation by expressing red or green fluorescent proteins in nuclei and conferring uridine/uracil or adenine auxotrophy to MAT1-1 and MAT1-2 strains of A. oryzae. The heterokaryon formation of A. oryzae was investigated in paired culture using the genetically modified strains. No sclerotial formation was observed in the hyphal contact regions of the two strains with the same auxotrophy, whereas numerous sclerotia were formed between the strains with different auxotrophies. In most of the formed sclerotia, the uridine/uracil and adenine auxotrophies were complemented, and both red and green fluorescence were detected, indicating that heterokaryotic fusants were formed by hyphal fusion before or during sclerotial formation. Moreover, overexpressing the sclR gene, which encodes a transcription factor promoting sclerotial formation, increased the number of heterokaryotic sclerotia formed between the two auxotrophic strains. Notably, these effects in sclerotial formation of heterokaryotic fusants were observed independently of the mating type pairing combinations. Taken together, these findings demonstrated that paring of different auxotrophs and sclR overexpression promote the formation of heterokaryotic sclerotia in A. oryzae.

Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein

NASA Astrophysics Data System (ADS)

Zosel, Franziska; Haenni, Dominik; Soranno, Andrea; Nettels, Daniel; Schuler, Benjamin

2017-10-01

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. To complement the information from FRET, we combine it with photoinduced electron transfer (PET) quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond time scale can be obtained reliably from a single set of measurements by the analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A more detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic molecular dynamics simulation of integrase. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of the dynamics and detailed mechanisms in unfolded and intrinsically disordered proteins.

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

DOE PAGES

Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; ...

2015-09-28

Elevated carbon dioxide (CO 2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO 2-induced stomatal movements by catalytic transmission of the CO 2 signal in guard cells. But, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO 2-regulated stomatal movements remains unknown. We express fluorescently tagged CAs in guard cells of ca1ca4 double-mutantmore » plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO 2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO 2 regulation of stomatal conductance. Mathematical modeling of cellular CO 2 catalysis suggests that the dynamics of the intracellular HCO 3 - concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Therefore, modeling supports the notion that the intracellular HCO 3 - concentration dynamics in guard cells are a key mechanism in mediating CO 2 -regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.« less

Wide-area mapping of resting state hemodynamic correlations at microvascular resolution with multi-contrast optical imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Senarathna, Janaka; Hadjiabadi, Darian; Gil, Stacy; Thakor, Nitish V.; Pathak, Arvind P.

2017-02-01

Different brain regions exhibit complex information processing even at rest. Therefore, assessing temporal correlations between regions permits task-free visualization of their `resting state connectivity'. Although functional MRI (fMRI) is widely used for mapping resting state connectivity in the human brain, it is not well suited for `microvascular scale' imaging in rodents because of its limited spatial resolution. Moreover, co-registered cerebral blood flow (CBF) and total hemoglobin (HbT) data are often unavailable in conventional fMRI experiments. Therefore, we built a customized system that combines laser speckle contrast imaging (LSCI), intrinsic optical signal (IOS) imaging and fluorescence imaging (FI) to generate multi-contrast functional connectivity maps at a spatial resolution of 10 μm. This system comprised of three illumination sources: a 632 nm HeNe laser (for LSCI), a 570 nm ± 5 nm filtered white light source (for IOS), and a 473 nm blue laser (for FI), as well as a sensitive CCD camera operating at 10 frames per second for image acquisition. The acquired data enabled visualization of changes in resting state neurophysiology at microvascular spatial scales. Moreover, concurrent mapping of CBF and HbT-based temporal correlations enabled in vivo mapping of how resting brain regions were linked in terms of their hemodynamics. Additionally, we complemented this approach by exploiting the transit times of a fluorescent tracer (Dextran-FITC) to distinguish arterial from venous perfusion. Overall, we demonstrated the feasibility of wide area mapping of resting state connectivity at microvascular resolution and created a new toolbox for interrogating neurovascular function.

Detection of membrane protein-protein interaction in planta based on dual-intein-coupled tripartite split-GFP association.

PubMed

Liu, Tzu-Yin; Chou, Wen-Chun; Chen, Wei-Yuan; Chu, Ching-Yi; Dai, Chen-Yi; Wu, Pei-Yu

2018-05-01

Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

An uncleaved signal peptide directs the Malus xiaojinensis iron transporter protein Mx IRT1 into the ER for the PM secretory pathway.

PubMed

Zhang, Peng; Tan, Song; Berry, James O; Li, Peng; Ren, Na; Li, Shuang; Yang, Guang; Wang, Wei-Bing; Qi, Xiao-Ting; Yin, Li-Ping

2014-11-07

Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1-29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake.

Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moosani, N.; Martin, R.H.

1994-09-01

Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomesmore » 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.« less

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes.

PubMed

Rouws, L F M; Meneses, C H S G; Guedes, H V; Vidal, M S; Baldani, J I; Schwab, S

2010-09-01

To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but beta-glucuronidase (GUS)-stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5-plant interaction. PAL5 could be isolated from the root surface (10(8) CFU g(-1)) and from surface-disinfected root and stem tissues (10(4) CFU g(-1)) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. These tools are of use to: (i) study PAL5 mutants affected in bacteria-plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.

Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides

PubMed Central

Zhou, Li Hong; Weizbauer, Renate A.; Singamaneni, Srikanth; Xu, Feng; Genin, Guy M.; Pickard, Barbara G.

2014-01-01

Background Certain membrane-associated arabinogalactan-proteins (AGPs) with lysine-rich sub-domains participate in plant growth, development and resistance to stress. To complement fluorescence imaging of such molecules when tagged and introduced transgenically to the cell periphery and to extend the groundwork for assessing molecular structure, some behaviours of surface-spread AGPs were visualized at the nanometre scale in a simplified electrostatic environment. Methods Enhanced green fluorescent protein (EGFP)-labelled LeAGP1 was isolated from Arabidopsis thaliana leaves using antibody-coated magnetic beads, deposited on graphite or mica, and examined with atomic force microscopy (AFM). Key Results When deposited at low concentration on graphite, LeAGP can form independent clusters and rings a few nanometres in diameter, often defining deep pits; the aperture of the rings depends on plating parameters. On mica, intermediate and high concentrations, respectively, yielded lacy meshes and solid sheets that could dynamically evolve arcs, rings, ‘pores’ and ‘co-pores’, and pits. Glucosyl Yariv reagent combined with the AGP to make very large and distinctive rings. Conclusions Diverse cell-specific nano-patterns of native lysine-rich AGPs are expected at the wall–membrane interface and, while there will not be an identical patterning in different environmental settings, AFM imaging suggests protein tendencies for surficial organization and thus opens new avenues for experimentation. Nanopore formation with Yariv reagents suggests how the reagent might bind with AGP to admit Ca2+ to cells and hints at ways in which AGP might be structured at some cell surfaces. PMID:25164699

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

Foetal Ureaplasma parvum bacteraemia as a function of gestation-dependent complement insufficiency: Evidence from a sheep model of pregnancy.

PubMed

Kemp, Matthew W; Ahmed, Shatha; Beeton, Michael L; Payne, Matthew S; Saito, Masatoshi; Miura, Yuichiro; Usuda, Haruo; Kallapur, Suhas G; Kramer, Boris W; Stock, Sarah J; Jobe, Alan H; Newnham, John P; Spiller, Owen B

2017-01-01

Complement is a central defence against sepsis, and increasing complement insufficiency in neonates of greater prematurity may predispose to increased sepsis. Ureaplasma spp. are the most frequently cultured bacteria from preterm blood samples. A sheep model of intrauterine Ureaplasma parvum infection was used to examine in vivo Ureaplasma bacteraemia at early and late gestational ages. Complement function and Ureaplasma killing assays were used to determine the correlation between complement potency and bactericidal activity of sera ex vivo. Ureaplasma was cultured from 50% of 95-day gestation lamb cord blood samples compared to 10% of 125-day gestation lambs. Bactericidal activity increased with increased gestational age, and a direct correlation between functional complement activity and bactericidal activity (R 2 =.86; P<.001) was found for 95-day gestational lambs. Ureaplasma bacteraemia in vivo was confined to early preterm lambs with low complement function, but Ureaplasma infection itself did not diminish complement levels. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Complement C5-inhibiting therapy for the thrombotic microangiopathies: accumulating evidence, but not a panacea

PubMed Central

Brocklebank, Vicky

2017-01-01

Abstract Thrombotic microangiopathy (TMA), characterized by organ injury occurring consequent to severe endothelial damage, can manifest in a diverse range of diseases. In complement-mediated atypical haemolytic uraemic syndrome (aHUS) a primary defect in complement, such as a mutation or autoantibody leading to over activation of the alternative pathway, predisposes to the development of disease, usually following exposure to an environmental trigger. The elucidation of the pathogenesis of aHUS resulted in the successful introduction of the complement inhibitor eculizumab into clinical practice. In other TMAs, although complement activation may be seen, its role in the pathogenesis remains to be confirmed by an interventional trial. Although many case reports in TMAs other than complement-mediated aHUS hint at efficacy, publication bias, concurrent therapies and in some cases the self-limiting nature of disease make broader interpretation difficult. In this article, we will review the evidence for the role of complement inhibition in complement-mediated aHUS and other TMAs. PMID:28980670

Theory of mind in SLI revisited: links with syntax, comparisons with ASD.

PubMed

Durrleman, Stephanie; Burnel, Morgane; Reboul, Anne

2017-11-01

According to the linguistic determinism approach, knowledge of sentential complements such as: John says that the earth is flat plays a crucial role in theory of mind (ToM) development by providing a means to represent explicitly people's mental attitudes and beliefs. This approach predicts that mastery of complements determines successful belief reasoning across explicit ToM tasks, even low-verbal ones, and across populations. (1) To investigate the link between a low-verbal ToM-task and complements in Specific Language Impairment (SLI), (2) To determine whether this population shows similar ToM performance to that of children with Autism Spectrum Disorder (ASD) or those with Typical Development (TD) once these groups are matched on competency for complements, (3) To explore whether complements conveying a falsehood without jeopardizing the veracity of the entire sentence, such as complements of verbs of communication, are more crucial for belief attribution than complements which do not have this property, namely complements of verbs of perception, (?John sees that the earth is flat). Children with SLI (n = 20), with ASD (n = 34) and TD (n = 30) completed sentence-picture-matching tasks assessing complementation with communication and perception verbs, as well as a picture-sequencing task assessing ToM. Children were furthermore evaluated for general grammatical and lexical abilities and non-verbal IQ. Results reveal that competency on complements relates to ToM performance with a low-verbal task in SLI, and that SLI, ASD and TD groups of equivalent performance on complements also perform similarly for ToM. Results further suggest that complements with an independent truth-value are the only ones to show a significant relation to ToM performance after teasing out the impact of non-verbal reasoning. This study suggests that clinical groups of different aetiologies as well as TD children perform comparably for ToM once they have similar complementation skills. Findings further highlight that specific types of complements, namely those with an independent truth value, relate in a special way to mentalizing. Future work should determine whether these specific structures could be effective in ToM remediation programmes. © 2017 Royal College of Speech and Language Therapists.

Targeting complement-mediated immunoregulation for cancer immunotherapy.

PubMed

Kolev, Martin; Markiewski, Maciej M

2018-06-01

Complement was initially discovered as an assembly of plasma proteins "complementing" the cytolytic activity of antibodies. However, our current knowledge places this complex system of several plasma proteins, receptors, and regulators in the center of innate immunity as a bridge between the initial innate responses and adaptive immune reactions. Consequently, complement appears to be pivotal for elimination of pathogens, not only as an early response defense, but by directing the subsequent adaptive immune response. The discovery of functional intracellular complement and its roles in cellular metabolism opened novel avenues for research and potential therapeutic implications. The recent studies demonstrating immunoregulatory functions of complement in the tumor microenvironment and the premetastatic niche shifted the paradigm on our understanding of functions of the complement system in regulating immunity. Several complement proteins, through their interaction with cells in the tumor microenvironment and in metastasis-targeted organs, contribute to modulating tumor growth, antitumor immunity, angiogenesis, and therefore, the overall progression of malignancy and, perhaps, responsiveness of cancer to different therapies. Here, we focus on recent progress in our understanding of immunostimulatory vs. immunoregulatory functions of complement and potential applications of these findings to the design of novel therapies for cancer patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

Complement activation promotes colitis-associated carcinogenesis through activating intestinal IL-1β/IL-17A axis.

PubMed

Ning, C; Li, Y-Y; Wang, Y; Han, G-C; Wang, R-X; Xiao, H; Li, X-Y; Hou, C-M; Ma, Y-F; Sheng, D-S; Shen, B-F; Feng, J-N; Guo, R-F; Li, Y; Chen, G-J

2015-11-01

Colitis-associated colorectal cancer (CAC) is the most serious complication of inflammatory bowel disease (IBD). Excessive complement activation has been shown to be involved in the pathogenesis of IBD. However, its role in the development of CAC is largely unknown. Here, using a CAC model induced by combined administration of azoxymethane (AOM) and dextran sulfate sodium (DSS), we demonstrated that complement activation was required for CAC pathogenesis. Deficiency in key components of complement (e.g., C3, C5, or C5a receptor) rendered tumor repression in mice subjected to AOM/DSS. Mechanistic investigation revealed that complement ablation dramatically reduced proinflammatory cytokine interleukin (IL)-1β levels in the colonic tissues that was mainly produced by infiltrating neutrophils. IL-1β promoted colon carcinogenesis by eliciting IL-17 response in intestinal myeloid cells. Furthermore, complement-activation product C5a represented a potent inducer for IL-1β in neutrophil, accounting for downregulation of IL-1β levels in the employed complement-deficient mice. Overall, our study proposes a protumorigenic role of complement in inflammation-related colorectal cancer and that the therapeutic strategies targeting complement may be beneficial for the treatment of CAC in clinic.

Protection of host cells by complement regulators.

PubMed

Schmidt, Christoph Q; Lambris, John D; Ricklin, Daniel

2016-11-01

The complement cascade is an ancient immune-surveillance system that not only provides protection from pathogen invasion but has also evolved to participate in physiological processes to maintain tissue homeostasis. The alternative pathway (AP) of complement activation is the evolutionarily oldest part of this innate immune cascade. It is unique in that it is continuously activated at a low level and arbitrarily probes foreign, modified-self, and also unaltered self-structures. This indiscriminate activation necessitates the presence of preformed regulators on autologous surfaces to spare self-cells from the undirected nature of AP activation. Although the other two canonical complement activation routes, the classical and lectin pathways, initiate the cascade more specifically through pattern recognition, their activity still needs to be tightly controlled to avoid excessive reactivity. It is the perpetual duty of complement regulators to protect the self from damage inflicted by inadequate complement activation. Here, we review the role of complement regulators as preformed mediators of defense, explain their common and specialized functions, and discuss selected cases in which alterations in complement regulators lead to disease. Finally, rational engineering approaches using natural complement inhibitors as potential therapeutics are highlighted. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Inactivation of complement by Loxosceles reclusa spider venom.

PubMed

Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

1979-07-01

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

The renaissance of complement therapeutics

PubMed Central

Ricklin, Daniel; Mastellos, Dimitrios C.; Reis, Edimara S.; Lambris, John D.

2018-01-01

The increasing number of clinical conditions that involve a pathological contribution from the complement system — many of which affect the kidneys — has spurred a regained interest in therapeutic options to modulate this host defence pathway. Molecular insight, technological advances, and the first decade of clinical experience with the complement-specific drug eculizumab, have contributed to a growing confidence in therapeutic complement inhibition. More than 20 candidate drugs that target various stages of the complement cascade are currently being evaluated in clinical trials, and additional agents are in preclinical development. Such diversity is clearly needed in view of the complex and distinct involvement of complement in a wide range of clinical conditions, including rare kidney disorders, transplant rejection and haemodialysis-induced inflammation. The existing drugs cannot be applied to all complement-driven diseases, and each indication has to be assessed individually. Alongside considerations concerning optimal points of intervention and economic factors, patient stratification will become essential to identify the best complement-specific therapy for each individual patient. This Review provides an overview of the therapeutic concepts, targets and candidate drugs, summarizes insights from clinical trials, and reflects on existing challenges for the development of complement therapeutics for kidney diseases and beyond. PMID:29199277

False Belief, Complementation Language, and Contextual Bias in Preschoolers

ERIC Educational Resources Information Center

Ng, Lisa; Cheung, Him; Xiao, Wen

2010-01-01

In the present study, we address two questions concerning the relation between children's false belief and their understanding of complex object complements. The first question is whether the previously demonstrated association between tensed complements and false belief generalizes to infinitival complements (de Villiers & Pyers, 2002). The…

The Syntax of Sentential Complementation in Turkish

ERIC Educational Resources Information Center

Predolac, Esra

2017-01-01

This dissertation examines primarily the syntactic, but also the semantic/pragmatic behavior of sentential complement clauses in Turkish and proposes a new classification of such complements. A head-final language, Turkish lacks an overt, lexical complementizer akin to English "that". The most frequent types of sentential complementation…

Complement activation in the tubulointerstitium: AKI, CKD, and in between.

PubMed

Brar, Jyoti E; Quigg, Richard J

2014-10-01

Complement activation is actively regulated to prevent injudicious activation, such as on peritubular endothelia and basolateral aspects of tubules. Miao et al. studied mice in which the key complement regulator, Crry, was deleted from tubular cells. This lacked functional consequence in unmanipulated animals. Yet, following ischemia-reperfusion, there was greater injury due to alternative pathway activation of C5. When the balance between complement activation and regulation is tipped towards the former, pathologic complement activation can ensue.

Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

PubMed Central

Li, Yujia; Parks, Griffith D.

2018-01-01

The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59. PMID:29693588

Complement System in Dermatological Diseases – Fire Under the Skin

PubMed Central

Panelius, Jaana; Meri, Seppo

2015-01-01

The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

Human L-ficolin, a recognition molecule of the lectin activation pathway of complement, activates complement by binding to pneumolysin, the major toxin of Streptococcus pneumoniae.

PubMed

Ali, Youssif M; Kenawy, Hany I; Muhammad, Adnan; Sim, Robert B; Andrew, Peter W; Schwaeble, Wilhelm J

2013-01-01

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

PubMed Central

Ali, Youssif M.; Kenawy, Hany I.; Muhammad, Adnan; Sim, Robert B.

2013-01-01

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum. PMID:24349316

Complement, a target for therapy in inflammatory and degenerative diseases.

PubMed

Morgan, B Paul; Harris, Claire L

2015-12-01

The complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. Complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. Drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. Only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. In this Review, we describe the history, current landscape and future directions for anti-complement therapies.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

In vitro C3 Deposition on Cryptococcus Capsule Occurs Via Multiple Complement Activation Pathways

PubMed Central

Mershon-Shier, Kileen L.; Vasuthasawat, Alex; Takahashi, Kazue; Morrison, Sherie L.; Beenhouwer, David O.

2011-01-01

Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and C. neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B−/− serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins. PMID:21723612

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5260 Complement C3b inactivator immunological test system. (a) Identification. A complement... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement C3b inactivator immunological test...

Complement Depletion Protects Lupus-prone Mice from Ischemia-reperfusion-initiated Organ Injury

DTIC Science & Technology

2012-10-25

injury, we sought to evaluate whether complement inhibition mitigates organ damage. We found that complement deple- tion with cobra venom factor... venom factor and C5a receptor antagonist were able to protect mice from local tissue damage, treatment with C5a receptor antagonist was not able to...Complement depletion or blockage of the complement pathway using molecules such as cobra venom factor (CVF) (24, 33) and C5a receptor antagonists (C5aRA

Effect of complement and its regulation on myasthenia gravis pathogenesis

PubMed Central

Kusner, Linda L; Kaminski, Henry J; Soltys, Jindrich

2015-01-01

Myasthenia gravis (MG) is primarily caused by antibodies directed towards the skeletal muscle acetylcholine receptor, leading to muscle weakness. Although these antibodies may induce compromise of neuromuscular transmission by blocking acetylcholine receptor function or antigenic modulation, the predominant mechanism of injury to the neuromuscular junction is complement-mediated lysis of the postsynaptic membrane. The vast majority of data to support the role of complement derives from experimentally acquired MG (EAMG). In this article, we review studies that demonstrate the central role of complement in EAMG and MG pathogenesis along with the emerging role of complement in T- and B-cell function, as well as the potential for complement inhibitor-based therapy to treat human MG. PMID:20477586

Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

PubMed

Hess, Christoph; Kemper, Claudia

2016-08-16

Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. Copyright © 2016 Elsevier Inc. All rights reserved.

The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea.

PubMed

Ren, Weichao; Liu, Na; Sang, Chengwei; Shi, Dongya; Zhou, Mingguo; Chen, Changjun; Qin, Qingming; Chen, Wenchan

2018-06-01

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8 , a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (Δ BcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the Δ BcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinerea IMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea . Copyright © 2018 American Society for Microbiology.

Non-Finite Complements in Russian, Serbian/Croatian, and Macedonian

ERIC Educational Resources Information Center

Kim, Bo Ra

2010-01-01

This study investigates the coherence properties of non-finite complements in Russian, Serbian/Croatian, and Macedonian. I demonstrate that Slavic non-finite complements do not project a uniform syntactic structure. The maximal projection of non-finite complements is not fixed but depends on the selectional properties of the matrix verb. I present…

The Production of Complement Clauses in Children with Language Impairment

ERIC Educational Resources Information Center

Steel, Gillian; Rose, Miranda; Eadie, Patricia

2016-01-01

Purpose: The purpose of this research was to provide a comprehensive description of complement-clause production in children with language impairment. Complement clauses were examined with respect to types of complement structure produced, verb use, and both semantic and syntactic accuracy. Method: A group of 17 children with language impairment…

Complement system biomarkers in epilepsy.

PubMed

Kopczynska, Maja; Zelek, Wioleta M; Vespa, Simone; Touchard, Samuel; Wardle, Mark; Loveless, Samantha; Thomas, Rhys H; Hamandi, Khalid; Morgan, B Paul

2018-05-24

To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study. Copyright © 2018. Published by Elsevier Ltd.

Complement in Non-Antibody-Mediated Kidney Diseases

PubMed Central

Angeletti, Andrea; Reyes-Bahamonde, Joselyn; Cravedi, Paolo; Campbell, Kirk N.

2017-01-01

The complement system is part of the innate immune response that plays important roles in protecting the host from foreign pathogens. The complement components and relative fragment deposition have long been recognized to be strongly involved also in the pathogenesis of autoantibody-related kidney glomerulopathies, leading to direct glomerular injury and recruitment of infiltrating inflammation pathways. More recently, unregulated complement activation has been shown to be associated with progression of non-antibody-mediated kidney diseases, including focal segmental glomerulosclerosis, C3 glomerular disease, thrombotic microangiopathies, or general fibrosis generation in progressive chronic kidney diseases. Some of the specific mechanisms associated with complement activation in these diseases were recently clarified, showing a dominant role of alternative activation pathway. Over the last decade, a growing number of anticomplement agents have been developed, and some of them are being approved for clinical use or already in use. Therefore, anticomplement therapies represent a realistic choice of therapeutic approaches for complement-related diseases. Herein, we review the complement system activation, regulatory mechanisms, their involvement in non-antibody-mediated glomerular diseases, and the recent advances in complement-targeting agents as potential therapeutic strategies. PMID:28748184

Role of Complement on Broken Surfaces After Trauma.

PubMed

Huber-Lang, Markus; Ignatius, Anita; Brenner, Rolf E

2015-01-01

Activation of both the complement and coagulation cascade after trauma and subsequent local and systemic inflammatory response represent a major scientific and clinical problem. After severe tissue injury and bone fracture, exposure of innate immunity to damaged cells and molecular debris is considered a main trigger of the posttraumatic danger response. However, the effects of cellular fragments (e.g., histones) on complement activation remain enigmatic. Furthermore, direct effects of "broken" bone and cartilage surfaces on the fluid phase response of complement and its interaction with key cells of connective tissues are still unknown. Here, we summarize data suggesting direct and indirect complement activation by extracellular and cellular danger associated molecular patterns. In addition, key complement components and the corresponding receptors (such as C3aR, C5aR) have been detected on "exposed surfaces" of the damaged regions. On a cellular level, multiple effects of complement activation products on osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells have been found.In conclusion, the complement system may be activated by trauma-altered surfaces and is crucially involved in connective tissue healing and posttraumatic systemic inflammatory response.

Progress and trends in complement therapeutics.

PubMed

Ricklin, Daniel; Lambris, John D

2013-01-01

The past few years have proven to be a highly successful and exciting period for the field of complement-directed drug discovery and development. Driven by promising experiences with the first marketed complement drugs, increased knowledge about the involvement of complement in health and disease, and improvements in structural and analytical techniques as well as animal models of disease, the field has seen a surge in creative approaches to therapeutically intervene at various stages of the cascade. An impressive panel of compounds that show promise in clinical trials is meanwhile being lined up in the pipelines of both small biotechnology and big pharmaceutical companies. Yet with this new focus on complement-targeted therapeutics, important questions concerning target selection, point and length of intervention, safety, and drug delivery emerge. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases and affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This chapter highlights the key changes in the field that shape our current perception of complement-targeted drugs and provides a brief overview of recent strategies and emerging trends. Selected examples of complement-related diseases and inhibitor classes are highlighted to illustrate the diversity and creativity in field.

Progress and Trends in Complement Therapeutics.

PubMed

Ricklin, Daniel; Lambris, John D

2013-01-01

The past few years have proven to be a highly successful and exciting period for the field of complement-directed drug discovery and development. Driven by promising experiences with the first marketed complement drugs, increased knowledge about the involvement of complement in health and disease, and improvements in structural and analytical techniques as well as animal models of disease, the field has seen a surge in creative approaches to therapeutically intervene at various stages of the cascade. An impressive panel of compounds that show promise in clinical trials is meanwhile being lined up in the pipelines of both small biotechnology and big pharmaceutical companies. Yet with this new focus on complement-targeted therapeutics, important questions concerning target selection, point and length of intervention, safety, and drug delivery emerge. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases and affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This chapter highlights the key changes in the field that shape our current perception of complement-targeted drugs and provides a brief overview of recent strategies and emerging trends. Selected examples of complement-related diseases and inhibitor classes are highlighted to illustrate the diversity and creativity in field.