Sample records for complex biochemical pathways

  1. Diversity of bile salts in fish and amphibians: evolution of a complex biochemical pathway.

    PubMed

    Hagey, Lee R; Møller, Peter R; Hofmann, Alan F; Krasowski, Matthew D

    2010-01-01

    Bile salts are the major end metabolites of cholesterol and are also important in lipid and protein digestion, as well as shaping of the gut microflora. Previous studies had demonstrated variation of bile salt structures across vertebrate species. We greatly extend prior surveys of bile salt variation in fish and amphibians, particularly in analysis of the biliary bile salts of Agnatha and Chondrichthyes. While there is significant structural variation of bile salts across all fish orders, bile salt profiles are generally stable within orders of fish and do not correlate with differences in diet. This large data set allowed us to infer evolutionary changes in the bile salt synthetic pathway. The hypothesized ancestral bile salt synthetic pathway, likely exemplified in extant hagfish, is simpler and much shorter than the pathway of most teleost fish and terrestrial vertebrates. Thus, the bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution. Analysis of the evolution of bile salt synthetic pathways provides a rich model system for the molecular evolution of a complex biochemical pathway in vertebrates.

  2. Computational multiscale modeling in protein--ligand docking.

    PubMed

    Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

    2009-01-01

    In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

  3. Simulation studies in biochemical signaling and enzyme reactions

    NASA Astrophysics Data System (ADS)

    Nelatury, Sudarshan R.; Vagula, Mary C.

    2014-06-01

    Biochemical pathways characterize various biochemical reaction schemes that involve a set of species and the manner in which they are connected. Determination of schematics that represent these pathways is an important task in understanding metabolism and signal transduction. Examples of these Pathways are: DNA and protein synthesis, and production of several macro-molecules essential for cell survival. A sustained feedback mechanism arises in gene expression and production of mRNA that lead to protein synthesis if the protein so synthesized serves as a transcription factor and becomes a repressor of the gene expression. The cellular regulations are carried out through biochemical networks consisting of reactions and regulatory proteins. Systems biology is a relatively new area that attempts to describe the biochemical pathways analytically and develop reliable mathematical models for the pathways. A complete understanding of chemical reaction kinetics is prohibitively hard thanks to the nonlinear and highly complex mechanisms that regulate protein formation, but attempting to numerically solve some of the governing differential equations seems to offer significant insight about their biochemical picture. To validate these models, one can perform simple experiments in the lab. This paper introduces fundamental ideas in biochemical signaling and attempts to take first steps into the understanding of biochemical oscillations. Initially, the two-pool model of calcium is used to describe the dynamics behind the oscillations. Later we present some elementary results showing biochemical oscillations arising from solving differential equations of Elowitz and Leibler using MATLAB software.

  4. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    PubMed

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-04

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

  5. Multiple Ion Binding Equilibria, Reaction Kinetics, and Thermodynamics in Dynamic Models of Biochemical Pathways

    PubMed Central

    Vinnakota, Kalyan C.; Wu, Fan; Kushmerick, Martin J.; Beard, Daniel A.

    2009-01-01

    The operation of biochemical systems in vivo and in vitro is strongly influenced by complex interactions between biochemical reactants and ions such as H+, Mg2+, K+, and Ca2+. These are important second messengers in metabolic and signaling pathways that directly influence the kinetics and thermodynamics of biochemical systems. Herein we describe the biophysical theory and computational methods to account for multiple ion binding to biochemical reactants and demonstrate the crucial effects of ion binding on biochemical reaction kinetics and thermodynamics. In simulations of realistic systems, the concentrations of these ions change with time due to dynamic buffering and competitive binding. In turn, the effective thermodynamic properties vary as functions of cation concentrations and important environmental variables such as temperature and overall ionic strength. Physically realistic simulations of biochemical systems require incorporating all of these phenomena into a coherent mathematical description. Several applications to physiological systems are demonstrated based on this coherent simulation framework. PMID:19216922

  6. Organization of biogeochemical nitrogen pathways with switch-like adjustment in fluctuating soil redox conditions

    PubMed Central

    Lamba, Sanjay; Bera, Soumen; Rashid, Mubasher; Medvinsky, Alexander B.; Acquisti, Claudia; Li, Bai-Lian

    2017-01-01

    Nitrogen is cycled throughout ecosystems by a suite of biogeochemical processes. The high complexity of the nitrogen cycle resides in an intricate interplay between reversible biochemical pathways alternatively and specifically activated in response to diverse environmental cues. Despite aggressive research, how the fundamental nitrogen biochemical processes are assembled and maintained in fluctuating soil redox conditions remains elusive. Here, we address this question using a kinetic modelling approach coupled with dynamical systems theory and microbial genomics. We show that alternative biochemical pathways play a key role in keeping nitrogen conversion and conservation properties invariant in fluctuating environments. Our results indicate that the biochemical network holds inherent adaptive capacity to stabilize ammonium and nitrate availability, and that the bistability in the formation of ammonium is linked to the transient upregulation of the amo-hao mediated nitrification pathway. The bistability is maintained by a pair of complementary subsystems acting as either source or sink type systems in response to soil redox fluctuations. It is further shown how elevated anthropogenic pressure has the potential to break down the stability of the system, altering substantially ammonium and nitrate availability in the soil, with dramatic effects on biodiversity. PMID:28280580

  7. The Curriculum Prerequisite Network: Modeling the Curriculum as a Complex System

    ERIC Educational Resources Information Center

    Aldrich, Preston R.

    2015-01-01

    This article advances the prerequisite network as a means to visualize the hidden structure in an academic curriculum. Networks have been used to represent a variety of complex systems ranging from social systems to biochemical pathways and protein interactions. Here, I treat the academic curriculum as a complex system with nodes representing…

  8. Toward structural elucidation of the gamma-secretase complex

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

    Li, H.; Wolfe, M. S.; Selkoe, D. J.

    2009-03-11

    {gamma}-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid {beta}-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge now is to illuminate the structure of this fascinating and important protease at atomic resolution. Here, we review recent progress on biochemical and biophysical probing of the structure of the four-component complex and discuss obstacles and potential pathways toward elucidating itsmore » detailed structure.« less

  9. The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder.

    PubMed

    Walden, Helen; Deans, Andrew J

    2014-01-01

    Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia, a disorder characterized by sensitivity to DNA interstrand crosslinking agents. The clinical features of cytopenia, developmental defects, and tumor predisposition are similar in each group, suggesting that the gene products participate in a common pathway. The Fanconi anemia DNA repair pathway consists of an anchor complex that recognizes damage caused by interstrand crosslinks, a multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several downstream repair proteins including nucleases and homologous recombination enzymes. We review progress in the use of structural and biochemical approaches to understanding how each FANC protein functions in this pathway.

  10. Current status of the use of modalities in wound care: electrical stimulation and ultrasound therapy.

    PubMed

    Ennis, William J; Lee, Claudia; Plummer, Malgorzata; Meneses, Patricio

    2011-01-01

    Wound healing is a complex pathway that requires cells, an appropriate biochemical environment (i.e., cytokines, chemokines), an extracellular matrix, perfusion, and the application of both macrostrain and microstrain. The process is both biochemically complex and energy dependent. Healing can be assisted in difficult cases through the use of physical modalities. In the current literature, there is much debate over which treatment modality, dosage level, and timing is optimal. The mechanism of action for both electrical stimulation and ultrasound are reviewed along with possible clinical applications for the plastic surgeon.

  11. The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

    PubMed

    Castelle, Cindy J; Roger, Magali; Bauzan, Marielle; Brugna, Myriam; Lignon, Sabrina; Nimtz, Manfred; Golyshina, Olga V; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2015-08-01

    The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Comparative Analysis of Argonaute-dependent Small RNA Pathways in Drosophila

    PubMed Central

    Zhou, Rui; Hotta, Ikuko; Denli, Ahmet M.; Hong, Pengyu; Perrimon, Norbert; Hannon, Gregory J.

    2008-01-01

    Summary The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes. PMID:19026789

  13. Biochemical Basis of Sestrin Physiological Activities

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

    Ho, Allison; Cho, Chun-Seok; Namkoong, Sim

    Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate agingmore » and age-associated diseases.« less

  14. [Fe-S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite.

    PubMed

    Charan, Manish; Choudhary, Hadi Hasan; Singh, Nidhi; Sadik, Mohammad; Siddiqi, Mohammad Imran; Mishra, Satish; Habib, Saman

    2017-08-01

    The relict plastid (apicoplast) of the malaria parasite is the site for important biochemical pathways and is essential for parasite survival. The sulfur mobilization (SUF) pathway of iron-sulfur [Fe-S] cluster assembly in the apicoplast of Plasmodium spp. is of interest due to its absence in the human host suggesting the possibility of antimalarial intervention through apicoplast [Fe-S] biogenesis. We report biochemical characterization of components of the Plasmodium falciparum apicoplast SUF pathway after the first step of SUF. In vitro interaction experiments and in vivo cross-linking showed that apicoplast-encoded PfSufB and apicoplast-targeted PfSufC and PfSufD formed a complex. The PfSufB-C 2 -D complex could function as a scaffold to assemble [4Fe-4S] clusters in vitro and activity of the PfSufC ATPase was enhanced by PfSufD. Two carrier proteins, the NifU-like protein PfNfu and the A-type carrier PfSufA are homodimers, the former mediating transfer of [4Fe-4S] from the scaffold to a model [4Fe-4S] target protein with higher efficiency. Conditional knockout of SufS, the enzyme catalyzing the first step of SUF, by selective excision in the mosquito stages of Plasmodium berghei severely impaired development of sporozoites in oocysts establishing essentiality of the SUF machinery in the vector. Our results delineate steps of the complete apicoplast SUF pathway and demonstrate its critical role in the parasite life cycle. © 2017 Federation of European Biochemical Societies.

  15. An algorithm for modularization of MAPK and calcium signaling pathways: comparative analysis among different species.

    PubMed

    Nayak, Losiana; De, Rajat K

    2007-12-01

    Signaling pathways are large complex biochemical networks. It is difficult to analyze the underlying mechanism of such networks as a whole. In the present article, we have proposed an algorithm for modularization of signal transduction pathways. Unlike studying a signaling pathway as a whole, this enables one to study the individual modules (less complex smaller units) easily and hence to study the entire pathway better. A comparative study of modules belonging to different species (for the same signaling pathway) has been made, which gives an overall idea about development of the signaling pathways over the taken set of species of calcium and MAPK signaling pathways. The superior performance, in terms of biological significance, of the proposed algorithm over an existing community finding algorithm of Newman [Newman MEJ. Modularity and community structure in networks. Proc Natl Acad Sci USA 2006;103(23):8577-82] has been demonstrated using the aforesaid pathways of H. sapiens.

  16. Hierarchical modularization of biochemical pathways using fuzzy-c means clustering.

    PubMed

    de Luis Balaguer, Maria A; Williams, Cranos M

    2014-08-01

    Biological systems that are representative of regulatory, metabolic, or signaling pathways can be highly complex. Mathematical models that describe such systems inherit this complexity. As a result, these models can often fail to provide a path toward the intuitive comprehension of these systems. More coarse information that allows a perceptive insight of the system is sometimes needed in combination with the model to understand control hierarchies or lower level functional relationships. In this paper, we present a method to identify relationships between components of dynamic models of biochemical pathways that reside in different functional groups. We find primary relationships and secondary relationships. The secondary relationships reveal connections that are present in the system, which current techniques that only identify primary relationships are unable to show. We also identify how relationships between components dynamically change over time. This results in a method that provides the hierarchy of the relationships among components, which can help us to understand the low level functional structure of the system and to elucidate potential hierarchical control. As a proof of concept, we apply the algorithm to the epidermal growth factor signal transduction pathway, and to the C3 photosynthesis pathway. We identify primary relationships among components that are in agreement with previous computational decomposition studies, and identify secondary relationships that uncover connections among components that current computational approaches were unable to reveal.

  17. Characterizing Strain Variation in Engineered E. coli Using a Multi-Omics-Based Workflow

    DOE PAGES

    Brunk, Elizabeth; George, Kevin W.; Alonso-Gutierrez, Jorge; ...

    2016-05-19

    Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proofmore » of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks.« less

  18. B-cell Ligand Processing Pathways Detected by Large-scale Comparative Analysis

    PubMed Central

    Towfic, Fadi; Gupta, Shakti; Honavar, Vasant; Subramaniam, Shankar

    2012-01-01

    The initiation of B-cell ligand recognition is a critical step for the generation of an immune response against foreign bodies. We sought to identify the biochemical pathways involved in the B-cell ligand recognition cascade and sets of ligands that trigger similar immunological responses. We utilized several comparative approaches to analyze the gene coexpression networks generated from a set of microarray experiments spanning 33 different ligands. First, we compared the degree distributions of the generated networks. Second, we utilized a pairwise network alignment algorithm, BiNA, to align the networks based on the hubs in the networks. Third, we aligned the networks based on a set of KEGG pathways. We summarized our results by constructing a consensus hierarchy of pathways that are involved in B cell ligand recognition. The resulting pathways were further validated through literature for their common physiological responses. Collectively, the results based on our comparative analyses of degree distributions, alignment of hubs, and alignment based on KEGG pathways provide a basis for molecular characterization of the immune response states of B-cells and demonstrate the power of comparative approaches (e.g., gene coexpression network alignment algorithms) in elucidating biochemical pathways involved in complex signaling events in cells. PMID:22917187

  19. Combining Flux Balance and Energy Balance Analysis for Large-Scale Metabolic Network: Biochemical Circuit Theory for Analysis of Large-Scale Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Beard, Daniel A.; Liang, Shou-Dan; Qian, Hong; Biegel, Bryan (Technical Monitor)

    2001-01-01

    Predicting behavior of large-scale biochemical metabolic networks represents one of the greatest challenges of bioinformatics and computational biology. Approaches, such as flux balance analysis (FBA), that account for the known stoichiometry of the reaction network while avoiding implementation of detailed reaction kinetics are perhaps the most promising tools for the analysis of large complex networks. As a step towards building a complete theory of biochemical circuit analysis, we introduce energy balance analysis (EBA), which compliments the FBA approach by introducing fundamental constraints based on the first and second laws of thermodynamics. Fluxes obtained with EBA are thermodynamically feasible and provide valuable insight into the activation and suppression of biochemical pathways.

  20. Identification of novel loci for the generation of reporter mice

    PubMed Central

    Rebecchi, Monica; Levandis, Giovanna

    2017-01-01

    Abstract Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress. PMID:27899606

  1. Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

    PubMed Central

    Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

    2018-01-01

    The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

  2. Congenital Chromosomal Syndromes—A Model for Pathogenesis

    PubMed Central

    Rohde, Russell A.

    1965-01-01

    The origin of anomalies in the chromosomal syndromes is believed to be both polyetiologic and polypathogenetic. Whereas some malformations quite clearly appear to result from single gene mutations or from genic imbalance due to individual monosomic or trisomic loci, other anomalies (singly or in complex patterns) are better interpreted as originating from disturbances in particular biochemical pathways which affect the development of a variety of traits. Additional phenogenetic studies and the use of sophisticated biochemical analysis in persons with complex patterns of anomalies should provide a truer understanding of disease mechanisms and provide guidance for future studies which are aimed at the treatment and prevention of these intriguing misadventures of Nature. PMID:5318572

  3. The JAK/STAT pathway in obesity and diabetes.

    PubMed

    Gurzov, Esteban N; Stanley, William J; Pappas, Evan G; Thomas, Helen E; Gough, Daniel J

    2016-08-01

    Diabetes mellitus are complex, multi-organ metabolic pathologies characterized by hyperglycemia. Emerging evidence shows that the highly conserved and potent JAK/STAT signaling pathway is required for normal homeostasis, and, when dysregulated, contributes to the development of obesity and diabetes. In this review, we analyze the role of JAK/STAT activation in the brain, liver, muscle, fat and pancreas, and how this affects the course of the disease. We also consider the therapeutic implications of targeting the JAK/STAT pathway in treatment of obesity and diabetes. © 2016 Federation of European Biochemical Societies.

  4. Viroid Pathogenicity: One Process, Many Faces

    PubMed Central

    Owens, Robert A.; Hammond, Rosemarie W.

    2009-01-01

    Despite the non-coding nature of their small RNA genomes, the visible symptoms of viroid infection resemble those associated with many plant virus diseases. Recent evidence indicates that viroid-derived small RNAs acting through host RNA silencing pathways play a key role in viroid pathogenicity. Host responses to viroid infection are complex, involving signaling cascades containing host-encoded protein kinases and crosstalk between hormonal and defense-signaling pathways. Studies of viroid-host interaction in the context of entire biochemical or developmental pathways are just beginning, and many working hypotheses have yet to be critically tested. PMID:21994551

  5. The underlying pathway structure of biochemical reaction networks

    PubMed Central

    Schilling, Christophe H.; Palsson, Bernhard O.

    1998-01-01

    Bioinformatics is yielding extensive, and in some cases complete, genetic and biochemical information about individual cell types and cellular processes, providing the composition of living cells and the molecular structure of its components. These components together perform integrated cellular functions that now need to be analyzed. In particular, the functional definition of biochemical pathways and their role in the context of the whole cell is lacking. In this study, we show how the mass balance constraints that govern the function of biochemical reaction networks lead to the translation of this problem into the realm of linear algebra. The functional capabilities of biochemical reaction networks, and thus the choices that cells can make, are reflected in the null space of their stoichiometric matrix. The null space is spanned by a finite number of basis vectors. We present an algorithm for the synthesis of a set of basis vectors for spanning the null space of the stoichiometric matrix, in which these basis vectors represent the underlying biochemical pathways that are fundamental to the corresponding biochemical reaction network. In other words, all possible flux distributions achievable by a defined set of biochemical reactions are represented by a linear combination of these basis pathways. These basis pathways thus represent the underlying pathway structure of the defined biochemical reaction network. This development is significant from a fundamental and conceptual standpoint because it yields a holistic definition of biochemical pathways in contrast to definitions that have arisen from the historical development of our knowledge about biochemical processes. Additionally, this new conceptual framework will be important in defining, characterizing, and studying biochemical pathways from the rapidly growing information on cellular function. PMID:9539712

  6. Adverse outcome pathways linked to population models as a methodology for investigating effects of chemical stressors

    EPA Science Inventory

    In addressing the complexity and toxicity of chemical contaminants in Great Lakes ecosystems, we describe an approach to link chemically induced alterations in molecular and biochemical endpoints to adverse outcomes in whole organisms and populations. Analysis of population impac...

  7. Classification of Chemical Compounds to Support Complex Queries in a Pathway Database

    PubMed Central

    Weidemann, Andreas; Kania, Renate; Peiss, Christian; Rojas, Isabel

    2004-01-01

    Data quality in biological databases has become a topic of great discussion. To provide high quality data and to deal with the vast amount of biochemical data, annotators and curators need to be supported by software that carries out part of their work in an (semi-) automatic manner. The detection of errors and inconsistencies is a part that requires the knowledge of domain experts, thus in most cases it is done manually, making it very expensive and time-consuming. This paper presents two tools to partially support the curation of data on biochemical pathways. The tool enables the automatic classification of chemical compounds based on their respective SMILES strings. Such classification allows the querying and visualization of biochemical reactions at different levels of abstraction, according to the level of detail at which the reaction participants are described. Chemical compounds can be classified in a flexible manner based on different criteria. The support of the process of data curation is provided by facilitating the detection of compounds that are identified as different but that are actually the same. This is also used to identify similar reactions and, in turn, pathways. PMID:18629066

  8. Prediction and Biochemical Demonstration of a Catabolic Pathway for the Osmoprotectant Proline Betaine

    PubMed Central

    Kumar, Ritesh; Zhao, Suwen; Vetting, Matthew W.; Wood, B. McKay; Sakai, Ayano; Cho, Kyuil; Solbiati, José; Almo, Steven C.; Sweedler, Jonathan V.; Jacobson, Matthew P.; Gerlt, John A.; Cronan, John E.

    2014-01-01

    ABSTRACT Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. PMID:24520058

  9. Training Signaling Pathway Maps to Biochemical Data with Constrained Fuzzy Logic: Quantitative Analysis of Liver Cell Responses to Inflammatory Stimuli

    PubMed Central

    Morris, Melody K.; Saez-Rodriguez, Julio; Clarke, David C.; Sorger, Peter K.; Lauffenburger, Douglas A.

    2011-01-01

    Predictive understanding of cell signaling network operation based on general prior knowledge but consistent with empirical data in a specific environmental context is a current challenge in computational biology. Recent work has demonstrated that Boolean logic can be used to create context-specific network models by training proteomic pathway maps to dedicated biochemical data; however, the Boolean formalism is restricted to characterizing protein species as either fully active or inactive. To advance beyond this limitation, we propose a novel form of fuzzy logic sufficiently flexible to model quantitative data but also sufficiently simple to efficiently construct models by training pathway maps on dedicated experimental measurements. Our new approach, termed constrained fuzzy logic (cFL), converts a prior knowledge network (obtained from literature or interactome databases) into a computable model that describes graded values of protein activation across multiple pathways. We train a cFL-converted network to experimental data describing hepatocytic protein activation by inflammatory cytokines and demonstrate the application of the resultant trained models for three important purposes: (a) generating experimentally testable biological hypotheses concerning pathway crosstalk, (b) establishing capability for quantitative prediction of protein activity, and (c) prediction and understanding of the cytokine release phenotypic response. Our methodology systematically and quantitatively trains a protein pathway map summarizing curated literature to context-specific biochemical data. This process generates a computable model yielding successful prediction of new test data and offering biological insight into complex datasets that are difficult to fully analyze by intuition alone. PMID:21408212

  10. Stable isotope probing to study functional components of complex microbial ecosystems.

    PubMed

    Mazard, Sophie; Schäfer, Hendrik

    2014-01-01

    This protocol presents a method of dissecting the DNA or RNA of key organisms involved in a specific biochemical process within a complex ecosystem. Stable isotope probing (SIP) allows the labelling and separation of nucleic acids from community members that are involved in important biochemical transformations, yet are often not the most numerically abundant members of a community. This pure culture-independent technique circumvents limitations of traditional microbial isolation techniques or data mining from large-scale whole-community metagenomic studies to tease out the identities and genomic repertoires of microorganisms participating in biological nutrient cycles. SIP experiments can be applied to virtually any ecosystem and biochemical pathway under investigation provided a suitable stable isotope substrate is available. This versatile methodology allows a wide range of analyses to be performed, from fatty-acid analyses, community structure and ecology studies, and targeted metagenomics involving nucleic acid sequencing. SIP experiments provide an effective alternative to large-scale whole-community metagenomic studies by specifically targeting the organisms or biochemical transformations of interest, thereby reducing the sequencing effort and time-consuming bioinformatics analyses of large datasets.

  11. Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase.

    PubMed

    Pazos, Elena; Vázquez, M Eugenio

    2014-02-01

    Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide-based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide-based luminescent peptide sensors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A defined role for multiple Fanconi anemia gene products in DNA-damage-associated ubiquitination.

    PubMed

    Tan, Winnie; Deans, Andrew J

    2017-06-01

    Fanconi anemia (FA) is an inherited blood disorder that causes bone marrow failure and high predisposition to cancers. The FA pathway guards the cell's genome stability by orchestrating the repair of interstrand cross-linking during the S phase of the cell cycle, preventing the chromosomal instability that is a key event in bone marrow failure syndrome. Central to the FA pathway is loss of monoubiquitinated forms of the Fanconi proteins FANCI and FANCD2, a process that is normally mediated by a "core complex" of seven other Fanconi proteins. Each protein, when mutated, can cause FA. The FA core-complex-catalyzed reaction is critical for signaling DNA cross-link damage such as that induced by chemotherapies. Here, we present a perspective on the current understanding of FANCI and FANCD2 monoubiquitination-mediated DNA repair. Our recent biochemical reconstitution of the monoubiquitination (and deubiquitination) reactions creates a paradigm for understanding FA. Further biochemical analysis will create new opportunities to address the leukemic phenotype of FA patients. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  13. Metabolic profiling reveals reprogramming of lipid metabolic pathways in treatment of polycystic ovary syndrome with 3-iodothyronamine.

    PubMed

    Selen Alpergin, Ebru S; Bolandnazar, Zeinab; Sabatini, Martina; Rogowski, Michael; Chiellini, Grazia; Zucchi, Riccardo; Assadi-Porter, Fariba M

    2017-01-01

    Complex diseases such as polycystic ovary syndrome (PCOS) are associated with intricate pathophysiological, hormonal, and metabolic feedbacks that make their early diagnosis challenging, thus increasing the prevalence risks for obesity, cardiovascular, and fatty liver diseases. To explore the crosstalk between endocrine and lipid metabolic pathways, we administered 3-iodothyronamine (T1AM), a natural analog of thyroid hormone, in a mouse model of PCOS and analyzed plasma and tissue extracts using multidisciplinary omics and biochemical approaches. T1AM administration induces a profound tissue-specific antilipogenic effect in liver and muscle by lowering gene expression of key regulators of lipid metabolism, PTP1B and PLIN2, significantly increasing metabolites (glucogenic, amino acids, carnitine, and citrate) levels, while enhancing protection against oxidative stress. In contrast, T1AM has an opposing effect on the regulation of estrogenic pathways in the ovary by upregulating STAR, CYP11A1, and CYP17A1. Biochemical measurements provide further evidence of significant reduction in liver cholesterol and triglycerides in post-T1AM treatment. Our results shed light onto tissue-specific metabolic vs. hormonal pathway interactions, thus illuminating the intricacies within the pathophysiology of PCOS This study opens up new avenues to design drugs for targeted therapeutics to improve quality of life in complex metabolic diseases. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  14. Quantitative RT-PCR Comparison of the Urea and Nitric Oxide Cycle Gene Transcripts in Adult Human Tissues

    PubMed Central

    Neill, Meaghan Anne; Aschner, Judy; Barr, Frederick; Summar, Marshall L.

    2009-01-01

    The urea cycle and nitric oxide cycle play significant roles in complex biochemical and physiologic reactions. These cycles have distinct biochemical goals including the clearance of waste nitrogen; the production of the intermediates ornithine, citrulline, and arginine for the urea cycle; and the production of nitric oxide for the nitric oxide pathway. Despite their disparate functions, the two pathways share two enzymes, argininosuccinic acid synthase and argininosuccinic acid lyase, and a transporter, citrin. Studying the gene expression of these enzymes is paramount in understanding these complex biochemical pathways. Here, we examine the expression of genes involved in the urea cycle and the nitric oxide cycle in a panel of eleven different tissue samples obtained from individual adults without known inborn errors of metabolism. In this study, the pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Our results show that these transcripts are differentially expressed in different tissues. The pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Using the co-expression profiles, we discovered that the combination of expression of enzyme transcripts as detected in our study, might serve to fulfill specific physiologic function(s) in tissue including urea production/nitrogen removal, arginine/citrulline production, nitric oxide production, and ornithine production. Our study reveals the importance of studying not only the expression profile of an enzyme of interest, but also studying the expression profiles of the other enzymes involved in a particular pathway so as to better understand the context of expression. The tissue patterns we observed highlight the variety of important functions they conduct and provide insight into many of the clinical observations from their disruption. PMID:19345634

  15. Evolution and the complexity of bacteriophages.

    PubMed

    Serwer, Philip

    2007-03-13

    The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages.

  16. The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

    PubMed Central

    Yamashita, Takayuki; Kupfer, Gary M.; Naf, Dieter; Suliman, Ahmed; Joenje, Hans; Asano, Shigetaka; D’Andrea, Alan D.

    1998-01-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway. PMID:9789045

  17. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

    PubMed Central

    Shao, Zengyi; Zhao, Hua; Zhao, Huimin

    2009-01-01

    The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional d-xylose utilization pathway (∼9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (∼11 kb DNA consisting of five genes) and a functional combined d-xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of eight genes) with high efficiencies (70–100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies. PMID:19074487

  18. Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway

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

    Hicks, Katherine A.; Ealick, Steven E.

    HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide furthermore » insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.« less

  19. The Fanconi anaemia pathway: new players and new functions.

    PubMed

    Ceccaldi, Raphael; Sarangi, Prabha; D'Andrea, Alan D

    2016-06-01

    The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance.

  20. Inflammation as a Therapeutic Target for Diabetic Neuropathies

    PubMed Central

    Ang, Lynn; Holmes, Crystal; Gallagher, Katherine; Feldman, Eva L.

    2016-01-01

    Diabetic neuropathies (DNs) are one of the most prevalent chronic complications of diabetes and a major cause of disability, high mortality, and poor quality of life. Given the complex anatomy of the peripheral nervous system and types of fiber dysfunction, DNs have a wide spectrum of clinical manifestations. The treatment of DNs continues to be challenging, likely due to the complex pathogenesis that involves an array of systemic and cellular imbalances in glucose and lipids metabolism. These lead to the activation of various biochemical pathways, including increased oxidative/nitrosative stress, activation of the polyol and protein kinase C pathways, activation of polyADP ribosylation, and activation of genes involved in neuronal damage, cyclooxygenase-2 activation, endothelial dysfunction, altered Na+/K+-ATPase pump function, impaired C-peptide-related signaling pathways, endoplasmic reticulum stress, and low-grade inflammation. This review summarizes current evidence regarding the role of low-grade inflammation as a potential therapeutic target for DNs. PMID:26897744

  1. Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

    PubMed Central

    Yu, Yang; Park, Ji-Won; Kwon, Hyun-Mi; Hwang, Hyun-Ok; Jang, In-Hwan; Masuda, Akiko; Kurokawa, Kenji; Nakayama, Hiroshi; Lee, Won-Jae; Dohmae, Naoshi; Zhang, Jinghai; Lee, Bok Luel

    2010-01-01

    In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects. PMID:20702416

  2. Analysis of DNA interactions using single-molecule force spectroscopy.

    PubMed

    Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

    2013-06-01

    Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

  3. The role of complex carbohydrate catabolism in the pathogenesis of invasive streptococci

    PubMed Central

    Shelburne, Samuel A.; Davenport, Michael T.; Keith, David B.; Musser, James M.

    2009-01-01

    Historically, the study of bacterial catabolism of complex carbohydrates has contributed to understanding basic bacterial physiology. Recently, however, genome-wide screens of streptococcal pathogenesis have identified genes encoding proteins involved in complex carbohydrate catabolism as participating in pathogen infectivity. Subsequent studies have focused on specific mechanisms by which carbohydrate utilization proteins might contribute to the ability of streptococci to colonize and infect the host. Moreover, transcriptome and biochemical analyses have uncovered novel regulatory pathways by which streptococci link environmental carbohydrate availability to virulence factor production. Herein we review new insights into the role of complex carbohydrates in streptococcal host-pathogen interaction. PMID:18508271

  4. Wound Healing: Biochemical Pathways and in vivo Studies.

    DTIC Science & Technology

    1980-02-01

    glycosaminoglycans (mucopolysaccharides) and glycoproteins (proteins with covalently bound hetero- polysaccharide chains). The matrix portion of the collagen unit is...the monosaccharides to the more complex mucopolysaccharides and glycoproteins and their role in the production and structure of collagen is evolving...glucosamine, and hexoses--glucose, galac- tose, and mannose. The monosaccharide pattern was similar in the wound tissue of the three species. These

  5. "Which Pathway Am I?" Using a Game Approach to Teach Students about Biochemical Pathways

    ERIC Educational Resources Information Center

    Ooi, Beng Guat; Sanger, Michael J.

    2009-01-01

    This game was designed to provide students with an alternative way to learn biochemical pathways through an interactive approach. In this game, students worked in pairs to help each other identify pathways taped to each other's backs by asking simple "yes or no" questions related to these pathways. This exercise was conducted after the traditional…

  6. Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

    PubMed

    Parisi, Federica; Riccardo, Sara; Daniel, Margaret; Saqcena, Mahesh; Kundu, Nandini; Pession, Annalisa; Grifoni, Daniela; Stocker, Hugo; Tabak, Esteban; Bellosta, Paola

    2011-09-27

    Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

  7. Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes

    PubMed Central

    Chuang, Jen-Zen; Vega, Carrie; Jun, Wenjin; Sung, Ching-Hwa

    2004-01-01

    Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations. PMID:15232620

  8. Deterministic modelling and stochastic simulation of biochemical pathways using MATLAB.

    PubMed

    Ullah, M; Schmidt, H; Cho, K H; Wolkenhauer, O

    2006-03-01

    The analysis of complex biochemical networks is conducted in two popular conceptual frameworks for modelling. The deterministic approach requires the solution of ordinary differential equations (ODEs, reaction rate equations) with concentrations as continuous state variables. The stochastic approach involves the simulation of differential-difference equations (chemical master equations, CMEs) with probabilities as variables. This is to generate counts of molecules for chemical species as realisations of random variables drawn from the probability distribution described by the CMEs. Although there are numerous tools available, many of them free, the modelling and simulation environment MATLAB is widely used in the physical and engineering sciences. We describe a collection of MATLAB functions to construct and solve ODEs for deterministic simulation and to implement realisations of CMEs for stochastic simulation using advanced MATLAB coding (Release 14). The program was successfully applied to pathway models from the literature for both cases. The results were compared to implementations using alternative tools for dynamic modelling and simulation of biochemical networks. The aim is to provide a concise set of MATLAB functions that encourage the experimentation with systems biology models. All the script files are available from www.sbi.uni-rostock.de/ publications_matlab-paper.html.

  9. The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

    PubMed

    Nolz, Jeffrey C; Gomez, Timothy S; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B; Shimizu, Yoji; Burkhardt, Janis K; Freedman, Bruce D; Billadeau, Daniel D

    2006-01-10

    The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

  10. Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

    PubMed Central

    Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

    2011-01-01

    Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

  11. Extending double modulation: combinatorial rules for identifying the modulations necessary for determining elasticities in metabolic pathways.

    PubMed

    Giersch, C; Cornish-Bowden, A

    1996-10-07

    The double modulation method for determining the elasticities of pathway enzymes, originally devised by Kacser & Burns (Biochem. Soc. Trans. 7, 1149-1160, 1979), is extended to pathways of complex topological structure, including branching and feedback loops. An explicit system of linear equations for the unknown elasticities is derived. The constraints imposed on this linear system imply that modulations of more than one enzyme are not necessarily independent. Simple combinatorial rules are described for identifying without using any algebra the set of independent modulations that allow the determination of the elasticities of any enzyme. By repeated application, the minimum numbers of modulations required to determine the elasticities of all enzymes of a given pathway can be determined. The procedure is illustrated with numerous examples.

  12. How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

    PubMed

    Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

    2017-03-15

    Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

  13. Classification and Analysis of Regulatory Pathways Using Graph Property, Biochemical and Physicochemical Property, and Functional Property

    PubMed Central

    Cai, Yu-Dong; Chou, Kuo-Chen

    2011-01-01

    Given a regulatory pathway system consisting of a set of proteins, can we predict which pathway class it belongs to? Such a problem is closely related to the biological function of the pathway in cells and hence is quite fundamental and essential in systems biology and proteomics. This is also an extremely difficult and challenging problem due to its complexity. To address this problem, a novel approach was developed that can be used to predict query pathways among the following six functional categories: (i) “Metabolism”, (ii) “Genetic Information Processing”, (iii) “Environmental Information Processing”, (iv) “Cellular Processes”, (v) “Organismal Systems”, and (vi) “Human Diseases”. The prediction method was established trough the following procedures: (i) according to the general form of pseudo amino acid composition (PseAAC), each of the pathways concerned is formulated as a 5570-D (dimensional) vector; (ii) each of components in the 5570-D vector was derived by a series of feature extractions from the pathway system according to its graphic property, biochemical and physicochemical property, as well as functional property; (iii) the minimum redundancy maximum relevance (mRMR) method was adopted to operate the prediction. A cross-validation by the jackknife test on a benchmark dataset consisting of 146 regulatory pathways indicated that an overall success rate of 78.8% was achieved by our method in identifying query pathways among the above six classes, indicating the outcome is quite promising and encouraging. To the best of our knowledge, the current study represents the first effort in attempting to identity the type of a pathway system or its biological function. It is anticipated that our report may stimulate a series of follow-up investigations in this new and challenging area. PMID:21980418

  14. Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

    PubMed Central

    McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

    2018-01-01

    Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

  15. Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

    2000-01-01

    PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

  16. Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

    PubMed

    Jain, Shruti; Bhattacharyya, Kausik; Bakshi, Rachit; Narang, Ankita; Brahmachari, Vani

    2017-04-01

    The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. The RNA-induced silencing complex: a versatile gene-silencing machine.

    PubMed

    Pratt, Ashley J; MacRae, Ian J

    2009-07-03

    RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. The ability of RISC to locate target RNAs has been co-opted by evolution many times to generate a broad spectrum of gene-silencing pathways. Here, we review the fundamental biochemical and biophysical properties of RISC that facilitate gene targeting and describe the various mechanisms of gene silencing known to exploit RISC activity.

  18. Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

    PubMed Central

    MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

    2008-01-01

    C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

  19. Modular decomposition of metabolic reaction networks based on flux analysis and pathway projection.

    PubMed

    Yoon, Jeongah; Si, Yaguang; Nolan, Ryan; Lee, Kyongbum

    2007-09-15

    The rational decomposition of biochemical networks into sub-structures has emerged as a useful approach to study the design of these complex systems. A biochemical network is characterized by an inhomogeneous connectivity distribution, which gives rise to several organizational features, including modularity. To what extent the connectivity-based modules reflect the functional organization of the network remains to be further explored. In this work, we examine the influence of physiological perturbations on the modular organization of cellular metabolism. Modules were characterized for two model systems, liver and adipocyte primary metabolism, by applying an algorithm for top-down partition of directed graphs with non-uniform edge weights. The weights were set by the engagement of the corresponding reactions as expressed by the flux distribution. For the base case of the fasted rat liver, three modules were found, carrying out the following biochemical transformations: ketone body production, glucose synthesis and transamination. This basic organization was further modified when different flux distributions were applied that describe the liver's metabolic response to whole body inflammation. For the fully mature adipocyte, only a single module was observed, integrating all of the major pathways needed for lipid storage. Weaker levels of integration between the pathways were found for the early stages of adipocyte differentiation. Our results underscore the inhomogeneous distribution of both connectivity and connection strengths, and suggest that global activity data such as the flux distribution can be used to study the organizational flexibility of cellular metabolism. Supplementary data are available at Bioinformatics online.

  20. Identification of Genetic Bases of Vibrio fluvialis Species-Specific Biochemical Pathways and Potential Virulence Factors by Comparative Genomic Analysis

    PubMed Central

    Lu, Xin; Liang, Weili; Wang, Yunduan; Xu, Jialiang

    2014-01-01

    Vibrio fluvialis is an important food-borne pathogen that causes diarrheal illness and sometimes extraintestinal infections in humans. In this study, we sequenced the genome of a clinical V. fluvialis strain and determined its phylogenetic relationships with other Vibrio species by comparative genomic analysis. We found that the closest relationship was between V. fluvialis and V. furnissii, followed by those with V. cholerae and V. mimicus. Moreover, based on genome comparisons and gene complementation experiments, we revealed genetic mechanisms of the biochemical tests that differentiate V. fluvialis from closely related species. Importantly, we identified a variety of genes encoding potential virulence factors, including multiple hemolysins, transcriptional regulators, and environmental survival and adaptation apparatuses, and the type VI secretion system, which is indicative of complex regulatory pathways modulating pathogenesis in this organism. The availability of V. fluvialis genome sequences may promote our understanding of pathogenic mechanisms for this emerging pathogen. PMID:24441165

  1. Model-Based Design of Biochemical Microreactors

    PubMed Central

    Elbinger, Tobias; Gahn, Markus; Neuss-Radu, Maria; Hante, Falk M.; Voll, Lars M.; Leugering, Günter; Knabner, Peter

    2016-01-01

    Mathematical modeling of biochemical pathways is an important resource in Synthetic Biology, as the predictive power of simulating synthetic pathways represents an important step in the design of synthetic metabolons. In this paper, we are concerned with the mathematical modeling, simulation, and optimization of metabolic processes in biochemical microreactors able to carry out enzymatic reactions and to exchange metabolites with their surrounding medium. The results of the reported modeling approach are incorporated in the design of the first microreactor prototypes that are under construction. These microreactors consist of compartments separated by membranes carrying specific transporters for the input of substrates and export of products. Inside the compartments of the reactor multienzyme complexes assembled on nano-beads by peptide adapters are used to carry out metabolic reactions. The spatially resolved mathematical model describing the ongoing processes consists of a system of diffusion equations together with boundary and initial conditions. The boundary conditions model the exchange of metabolites with the neighboring compartments and the reactions at the surface of the nano-beads carrying the multienzyme complexes. Efficient and accurate approaches for numerical simulation of the mathematical model and for optimal design of the microreactor are developed. As a proof-of-concept scenario, a synthetic pathway for the conversion of sucrose to glucose-6-phosphate (G6P) was chosen. In this context, the mathematical model is employed to compute the spatio-temporal distributions of the metabolite concentrations, as well as application relevant quantities like the outflow rate of G6P. These computations are performed for different scenarios, where the number of beads as well as their loading capacity are varied. The computed metabolite distributions show spatial patterns, which differ for different experimental arrangements. Furthermore, the total output of G6P increases for scenarios where microcompartimentation of enzymes occurs. These results show that spatially resolved models are needed in the description of the conversion processes. Finally, the enzyme stoichiometry on the nano-beads is determined, which maximizes the production of glucose-6-phosphate. PMID:26913283

  2. Modular and Stochastic Approaches to Molecular Pathway Models of ATM, TGF beta, and WNT Signaling

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; O'Neill, Peter; Ponomarev, Artem; Carra, Claudio; Whalen, Mary; Pluth, Janice M.

    2009-01-01

    Deterministic pathway models that describe the biochemical interactions of a group of related proteins, their complexes, activation through kinase, etc. are often the basis for many systems biology models. Low dose radiation effects present a unique set of challenges to these models including the importance of stochastic effects due to the nature of radiation tracks and small number of molecules activated, and the search for infrequent events that contribute to cancer risks. We have been studying models of the ATM, TGF -Smad and WNT signaling pathways with the goal of applying pathway models to the investigation of low dose radiation cancer risks. Modeling challenges include introduction of stochastic models of radiation tracks, their relationships to more than one substrate species that perturb pathways, and the identification of a representative set of enzymes that act on the dominant substrates. Because several pathways are activated concurrently by radiation the development of modular pathway approach is of interest.

  3. Biochemical-Pathway Diversity in Archaebacteria

    DTIC Science & Technology

    1990-08-30

    Classification) (U) Biochemical-pathway diversity in Archaebacteria 12 PERSONAL AUTHOR(S) I Jensen, Roy-A. i3o. TYPE OF REN" RT 12b. Tki~ 0’E D-30-9 4...by block numtb.sj FIEL I ROU I SIGRLJP Archaebacteria , biochemical diversity, prephenate 06 03. 1 dehydratase, aromatic amino acid biosynthesis t...1988 RE10SE: lo assess the extent to which the archaebacteria possess unique biochemical features of aromatic amino acid biosynthesis and regulation and

  4. TGF-β control of stem cell differentiation genes.

    PubMed

    Massagué, Joan; Xi, Qiaoran

    2012-07-04

    The canonical TGF-β/Smad signaling pathway was delineated in the mid 90s and enriched over the past decade with many findings about its specificity, regulation, networking, and malfunctions in disease. However, a growing understanding of the chromatin status of a critical class of TGF-β target genes - the genes controlling differentiation of embryonic stem cells - recently prompted a reexamination of this pathway and its critical role in the regulation of stem cell differentiation. The new findings reveal master regulators of the pluripotent state set the stage for Smad-mediated activation of master regulators of the next differentiation stage. Furthermore, a novel branch of the TGF-β/Smad pathway has been identified in which a chromatin-reading Smad complex makes the master differentiation genes accessible to canonical Smad complexes for transcriptional activation. These findings provide exciting new insights into the global role of TGF-β signaling in the regulators of stem cell fate. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Juxtaposed Polycomb complexes co-regulate vertebral identity.

    PubMed

    Kim, Se Young; Paylor, Suzanne W; Magnuson, Terry; Schumacher, Armin

    2006-12-01

    Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

  6. Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching

    NASA Astrophysics Data System (ADS)

    Kraft, Lewis J.; Kenworthy, Anne K.

    2012-01-01

    The protein microtubule-associated protein 1, light chain 3 (LC3) functions in autophagosome formation and plays a central role in the autophagy pathway. Previously, we found LC3 diffuses more slowly in cells than is expected for a freely diffusing monomer, suggesting it may constitutively associate with a macromolecular complex containing other protein components of the pathway. In the current study, we used Förster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4BC74A, a catalytically inactive mutant of the cysteine protease involved in lipidation and de-lipidation of LC3, as a model system to probe protein complex formation in the autophagy pathway. We show Atg4BC74A is in FRET proximity with LC3 in both the cytoplasm and nucleus of living cells, consistent with previous biochemical evidence that suggests these proteins directly interact. In addition, overexpressed Atg4BC74A diffuses significantly more slowly than predicted based on its molecular weight, and its translational diffusion coefficient is significantly slowed upon coexpression with LC3 to match that of LC3 itself. Taken together, these results suggest Atg4BC74A and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells.

  7. Biophysical and biochemical constraints imposed by salt stress: learning from halophytes

    PubMed Central

    Duarte, Bernardo; Sleimi, Noomene; Caçador, Isabel

    2014-01-01

    Soil salinization is one of the most important factors impacting plant productivity. About 3.6 billion of the world’s 5.2 billion ha of agricultural dry land, have already suffered erosion, degradation, and salinization. Halophytes are typically considered as plants able to complete their life cycle in environments where the salt concentration is above 200 mM NaCl. Salinity adjustment is a complex phenomenon but essential mechanism to overcome salt stress, with both biophysical and biochemical implications. At this level, halophytes evolved in several directions, adopting different strategies. Otherwise, the lack of adaptation to a salt environment would negatively affect their electron transduction pathways and the entire energetic metabolism, the foundation of every plant photosynthesis and biomass production. The maintenance of ionic homeostasis is in the basis of all cellular counteractive measures, in particular in terms of redox potential and energy transduction. In the present work the biophysical mechanisms underlying energy capture and transduction in halophytes are discussed alongside with their relation with biochemical counteractive mechanisms, integrating data from photosynthetic light harvesting complexes, electron transport chains to the quinone pools, carbon fixation, and energy dissipation metabolism. PMID:25566311

  8. The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

    PubMed Central

    Nolz, Jeffrey C.; Gomez, Timothy S.; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B.; Shimizu, Yoji; Burkhardt, Janis K.; Freedman, Bruce D.; Billadeau, Daniel D.

    2007-01-01

    Summary Background The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. Results By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and β-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCγ1 activation and IP3-mediated store release. Conclusions These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation. PMID:16401421

  9. [Fanconi anemia: cellular and molecular features].

    PubMed

    Macé, G; Briot, D; Guervilly, J-H; Rosselli, F

    2007-02-01

    Fanconi anemia (FA) is a recessive human cancer prone syndrome featuring bone marrow failure, developmental abnormalities and hypersensitivity to DNA crosslinking agents exposure. 11 among 12 FA gene have been isolated. The biochemical functions of the FANC proteins remain poorly understood. Anyhow, to cope with DNA crosslinks a cell needs a functional FANC pathway. Moreover, the FANC proteins appear to be involved in cell protection against oxidative damage and in the control of TNF-alpha activity. In this review, we describe the current understanding of the FANC pathway and we present how it may be integrated in the complex networks of proteins involved in maintaining the cellular homeostasis.

  10. Programmable chemical reaction networks: emulating regulatory functions in living cells using a bottom-up approach.

    PubMed

    van Roekel, Hendrik W H; Rosier, Bas J H M; Meijer, Lenny H H; Hilbers, Peter A J; Markvoort, Albert J; Huck, Wilhelm T S; de Greef, Tom F A

    2015-11-07

    Living cells are able to produce a wide variety of biological responses when subjected to biochemical stimuli. It has become apparent that these biological responses are regulated by complex chemical reaction networks (CRNs). Unravelling the function of these circuits is a key topic of both systems biology and synthetic biology. Recent progress at the interface of chemistry and biology together with the realisation that current experimental tools are insufficient to quantitatively understand the molecular logic of pathways inside living cells has triggered renewed interest in the bottom-up development of CRNs. This builds upon earlier work of physical chemists who extensively studied inorganic CRNs and showed how a system of chemical reactions can give rise to complex spatiotemporal responses such as oscillations and pattern formation. Using purified biochemical components, in vitro synthetic biologists have started to engineer simplified model systems with the goal of mimicking biological responses of intracellular circuits. Emulation and reconstruction of system-level properties of intracellular networks using simplified circuits are able to reveal key design principles and molecular programs that underlie the biological function of interest. In this Tutorial Review, we present an accessible overview of this emerging field starting with key studies on inorganic CRNs followed by a discussion of recent work involving purified biochemical components. Finally, we review recent work showing the versatility of programmable biochemical reaction networks (BRNs) in analytical and diagnostic applications.

  11. Ran1 functions to control the Cdc10/Sct1 complex through Puc1.

    PubMed Central

    Caligiuri, M; Connolly, T; Beach, D

    1997-01-01

    We have undertaken a biochemical analysis of the regulation of the G1/S-phase transition and commitment to the cell cycle in the fission yeast Schizosaccharomyces pombe. The execution of Start requires the activity of the Cdc2 protein kinase and the Sct1/Cdc10 transcription complex. Progression through G1 also requires the Ran1 protein kinase whose inactivation leads to activation of the meiotic pathway under conditions normally inhibitory to this process. We have found that in addition to Cdc2, Sct1/Cdc10 complex formation requires Ran1. We demonstrate that the Puc1 cyclin associates with Ran1 and Cdc10 in vivo and that the Ran1 protein kinase functions to control the association between Puc1 and Cdc10. In addition, we present evidence that the phosphorylation state of Cdc10 is altered upon inactivation of Ran1. These results provide biochemical evidence that demonstrate one mechanism by which the Ran1 protein kinase serves to control cell fate through Cdc10 and Puc1. Images PMID:9201720

  12. Native gel analysis for RISC assembly.

    PubMed

    Kawamata, Tomoko; Tomari, Yukihide

    2011-01-01

    Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

  13. Conserved and species-specific molecular denominators in mammalian skeletal muscle aging.

    PubMed

    Mercken, Evi M; Capri, Miriam; Carboneau, Bethany A; Conte, Maria; Heidler, Juliana; Santoro, Aurelia; Martin-Montalvo, Alejandro; Gonzalez-Freire, Marta; Khraiwesh, Husam; González-Reyes, José A; Moaddel, Ruin; Zhang, Yongqing; Becker, Kevin G; Villalba, José M; Mattison, Julie A; Wittig, Ilka; Franceschi, Claudio; de Cabo, Rafael

    2017-01-01

    Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan (young, middle, and old) to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress (as assessed by protein carbonyl adducts and 4-hydroxynonenal) is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process.

  14. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino sugar 13C analysis showed that oxidizing catabolic pathways and anabolic pathways, i.e. building-up new cellular compounds, occurred in soils simultaneously. This involved an intensive C recycling within the microorganisms that was observed not only for cytosolic compounds but also for cell wall polymers. Fungal metabolism and fluxes were slower than bacterial intracellular C recycling and turnover. Furthermore, position-specific labeling of glutamate and subsequent 13C analysis of microbial phospholipid fatty acids (PLFA) revealed starvation pathways, which were only active in specific microbial groups in soils. These studies revealed that position-specific labeling enables the reconstruction of metabolic pathways of LMWOS within diverse microbial communities in complex media such as soil. Processes occurring simultaneously in soil i.e. 1) within individual, reversible metabolic pathways and 2) in various microbial groups could be traced by position-specific labeling in soils in situ. Tracing these pathways and understanding their regulating factors are crucial for soil C fluxomics, the extremely complex network of transformations towards mineralization versus the formation of microbial biomass compounds. Quantitative models to assess microbial group specific metabolic networks can be generated and parameterized by this approach. The submolecular knowledge of transformation steps and biochemical pathways in soils and their regulating factors is essential for understanding C cycling and long-term C storage in soils.

  15. Developing optimal input design strategies in cancer systems biology with applications to microfluidic device engineering.

    PubMed

    Menolascina, Filippo; Bellomo, Domenico; Maiwald, Thomas; Bevilacqua, Vitoantonio; Ciminelli, Caterina; Paradiso, Angelo; Tommasi, Stefania

    2009-10-15

    Mechanistic models are becoming more and more popular in Systems Biology; identification and control of models underlying biochemical pathways of interest in oncology is a primary goal in this field. Unfortunately the scarce availability of data still limits our understanding of the intrinsic characteristics of complex pathologies like cancer: acquiring information for a system understanding of complex reaction networks is time consuming and expensive. Stimulus response experiments (SRE) have been used to gain a deeper insight into the details of biochemical mechanisms underlying cell life and functioning. Optimisation of the input time-profile, however, still remains a major area of research due to the complexity of the problem and its relevance for the task of information retrieval in systems biology-related experiments. We have addressed the problem of quantifying the information associated to an experiment using the Fisher Information Matrix and we have proposed an optimal experimental design strategy based on evolutionary algorithm to cope with the problem of information gathering in Systems Biology. On the basis of the theoretical results obtained in the field of control systems theory, we have studied the dynamical properties of the signals to be used in cell stimulation. The results of this study have been used to develop a microfluidic device for the automation of the process of cell stimulation for system identification. We have applied the proposed approach to the Epidermal Growth Factor Receptor pathway and we observed that it minimises the amount of parametric uncertainty associated to the identified model. A statistical framework based on Monte-Carlo estimations of the uncertainty ellipsoid confirmed the superiority of optimally designed experiments over canonical inputs. The proposed approach can be easily extended to multiobjective formulations that can also take advantage of identifiability analysis. Moreover, the availability of fully automated microfluidic platforms explicitly developed for the task of biochemical model identification will hopefully reduce the effects of the 'data rich--data poor' paradox in Systems Biology.

  16. Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways.

    PubMed

    Shambharkar, Prashant B; Blonska, Marzenna; Pappu, Bhanu P; Li, Hongxiu; You, Yun; Sakurai, Hiroaki; Darnay, Bryant G; Hara, Hiromitsu; Penninger, Josef; Lin, Xin

    2007-04-04

    The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

  17. Modeling of the U1 snRNP assembly pathway in alternative splicing in human cells using Petri nets.

    PubMed

    Kielbassa, J; Bortfeldt, R; Schuster, S; Koch, I

    2009-02-01

    The investigation of spliceosomal processes is currently a topic of intense research in molecular biology. In the molecular mechanism of alternative splicing, a multi-protein-RNA complex - the spliceosome - plays a crucial role. To understand the biological processes of alternative splicing, it is essential to comprehend the biogenesis of the spliceosome. In this paper, we propose the first abstract model of the regulatory assembly pathway of the human spliceosomal subunit U1. Using Petri nets, we describe its highly ordered assembly that takes place in a stepwise manner. Petri net theory represents a mathematical formalism to model and analyze systems with concurrent processes at different abstraction levels with the possibility to combine them into a uniform description language. There exist many approaches to determine static and dynamic properties of Petri nets, which can be applied to analyze biochemical systems. In addition, Petri net tools usually provide intuitively understandable graphical network representations, which facilitate the dialog between experimentalists and theoreticians. Our Petri net model covers binding, transport, signaling, and covalent modification processes. Through the computation of structural and behavioral Petri net properties and their interpretation in biological terms, we validate our model and use it to get a better understanding of the complex processes of the assembly pathway. We can explain the basic network behavior, using minimal T-invariants which represent special pathways through the network. We find linear as well as cyclic pathways. We determine the P-invariants that represent conserved moieties in a network. The simulation of the net demonstrates the importance of the stability of complexes during the maturation pathway. We can show that complexes that dissociate too fast, hinder the formation of the complete U1 snRNP.

  18. Cellular compartmentalization of secondary metabolism

    PubMed Central

    Kistler, H. Corby; Broz, Karen

    2015-01-01

    Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g., amino acids, acetyl CoA, NADPH), enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported. PMID:25709603

  19. Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W(gm)ZOflL.

    PubMed

    Krnáčová, Katarína; Rýdlová, Ivana; Vinarčíková, Michaela; Krajčovič, Juraj; Vesteg, Matej; Horváth, Anton

    2015-03-12

    The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. Consensus-phenotype integration of transcriptomic and metabolomic data implies a role for metabolism in the chemosensitivity of tumour cells.

    PubMed

    Cavill, Rachel; Kamburov, Atanas; Ellis, James K; Athersuch, Toby J; Blagrove, Marcus S C; Herwig, Ralf; Ebbels, Timothy M D; Keun, Hector C

    2011-03-01

    Using transcriptomic and metabolomic measurements from the NCI60 cell line panel, together with a novel approach to integration of molecular profile data, we show that the biochemical pathways associated with tumour cell chemosensitivity to platinum-based drugs are highly coincident, i.e. they describe a consensus phenotype. Direct integration of metabolome and transcriptome data at the point of pathway analysis improved the detection of consensus pathways by 76%, and revealed associations between platinum sensitivity and several metabolic pathways that were not visible from transcriptome analysis alone. These pathways included the TCA cycle and pyruvate metabolism, lipoprotein uptake and nucleotide synthesis by both salvage and de novo pathways. Extending the approach across a wide panel of chemotherapeutics, we confirmed the specificity of the metabolic pathway associations to platinum sensitivity. We conclude that metabolic phenotyping could play a role in predicting response to platinum chemotherapy and that consensus-phenotype integration of molecular profiling data is a powerful and versatile tool for both biomarker discovery and for exploring the complex relationships between biological pathways and drug response.

  1. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  2. Protein design for pathway engineering.

    PubMed

    Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Regulation of metabolism by the Mediator complex.

    PubMed

    Youn, Dou Yeon; Xiaoli, Alus M; Pessin, Jeffrey E; Yang, Fajun

    2016-01-01

    The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its best-known function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the context-dependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.

  4. Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli.

    PubMed

    Zhang, Congqiang; Seow, Vui Yin; Chen, Xixian; Too, Heng-Phon

    2018-05-11

    Optimization of metabolic pathways consisting of large number of genes is challenging. Multivariate modular methods (MMMs) are currently available solutions, in which reduced regulatory complexities are achieved by grouping multiple genes into modules. However, these methods work well for balancing the inter-modules but not intra-modules. In addition, application of MMMs to the 15-step heterologous route of astaxanthin biosynthesis has met with limited success. Here, we expand the solution space of MMMs and develop a multidimensional heuristic process (MHP). MHP can simultaneously balance different modules by varying promoter strength and coordinating intra-module activities by using ribosome binding sites (RBSs) and enzyme variants. Consequently, MHP increases enantiopure 3S,3'S-astaxanthin production to 184 mg l -1 day -1 or 320 mg l -1 . Similarly, MHP improves the yields of nerolidol and linalool. MHP may be useful for optimizing other complex biochemical pathways.

  5. Sulfoquinovose in the biosphere: occurrence, metabolism and functions.

    PubMed

    Goddard-Borger, Ethan D; Williams, Spencer J

    2017-02-20

    The sulfonated carbohydrate sulfoquinovose (SQ) is produced in quantities estimated at some 10 billion tonnes annually and is thus a major participant in the global sulfur biocycle. SQ is produced by most photosynthetic organisms and incorporated into the sulfolipid sulfoquinovosyl diacylglycerol (SQDG), as well as within some archaea for incorporation into glycoprotein N-glycans. SQDG is found mainly within the thylakoid membranes of the chloroplast, where it appears to be important for membrane structure and function and for optimal activity of photosynthetic protein complexes. SQDG metabolism within the sulfur cycle involves complex biosynthetic and catabolic processes. SQDG biosynthesis is largely conserved within plants, algae and bacteria. On the other hand, two major sulfoglycolytic pathways have been discovered for SQDG degradation, the sulfo-Embden-Meyerhof-Parnas (sulfo-EMP) and sulfo-Entner-Doudoroff (sulfo-ED) pathways, which mirror the major steps in the glycolytic EMP and ED pathways. Sulfoglycolysis produces C3-sulfonates, which undergo biomineralization to inorganic sulfur species, completing the sulfur cycle. This review discusses the discovery and structural elucidation of SQDG and archaeal N-glycans, the occurrence, distribution, and speciation of SQDG, and metabolic pathways leading to the biosynthesis of SQDG and its catabolism through sulfoglycolytic and biomineralization pathways to inorganic sulfur. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair.

    PubMed

    Leung, Justin Wai Chung; Wang, Yucai; Fong, Ka Wing; Huen, Michael Shing Yan; Li, Lei; Chen, Junjie

    2012-03-20

    The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway.

  7. Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair

    PubMed Central

    Leung, Justin Wai Chung; Wang, Yucai; Fong, Ka Wing; Huen, Michael Shing Yan; Li, Lei; Chen, Junjie

    2012-01-01

    The Fanconi anemia (FA) pathway participates in interstrand cross-link (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemia-associated proteins (FAAP24 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of cross-linked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP20, which contains a RAD18-like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP20 promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP20 displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway. PMID:22396592

  8. Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

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

    Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

    In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradationmore » pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.« less

  9. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  10. Ligand Receptor-Mediated Regulation of Growth in Plants.

    PubMed

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase family (approximately 5% of the protein coding genes), although the specific function for only a few dozen of these kinases is clearly established. Recent comparative genomics studies have revealed that parasitic nematodes and pathogenic microbes produce plant peptide hormone mimics that target specific plant plasma membrane receptor-like protein kinases, thus usurping endogenous signaling pathways for their own pathogenic purposes. With biochemical, genetic, and physiological analyses of the regulation of hormone receptor signal pathways, we are thus just now beginning to understand how plants optimize the development of their body shape and cope with constantly changing environmental conditions. © 2017 Elsevier Inc. All rights reserved.

  11. Post-translational modifications are key players of the Legionella pneumophila infection strategy

    PubMed Central

    Michard, Céline; Doublet, Patricia

    2015-01-01

    Post-translational modifications (PTMs) are widely used by eukaryotes to control the enzymatic activity, localization or stability of their proteins. Traditionally, it was believed that the broad biochemical diversity of the PTMs is restricted to eukaryotic cells, which exploit it in extensive networks to fine-tune various and complex cellular functions. During the last decade, the advanced detection methods of PTMs and functional studies of the host–pathogen relationships highlight that bacteria have also developed a large arsenal of PTMs, particularly to subvert host cell pathways to their benefit. Legionella pneumophila, the etiological agent of the severe pneumonia legionellosis, is the paradigm of highly adapted intravacuolar pathogens that have set up sophisticated biochemical strategies. Among them, L. pneumophila has evolved eukaryotic-like and rare/novel PTMs to hijack host cell processes. Here, we review recent progress about the diversity of PTMs catalyzed by Legionella: ubiquitination, prenylation, phosphorylation, glycosylation, methylation, AMPylation, and de-AMPylation, phosphocholination, and de-phosphocholination. We focus on the host cell pathways targeted by the bacteria catalyzed PTMs and we stress the importance of the PTMs in the Legionella infection strategy. Finally, we highlight that the discovery of these PTMs undoubtedly made significant breakthroughs on the molecular basis of Legionella pathogenesis but also lead the way in improving our knowledge of the eukaryotic PTMs and complex cellular processes that are associated to. PMID:25713573

  12. Following Carbon Isotopes from Methane to Molecules

    NASA Astrophysics Data System (ADS)

    Freeman, K. H.

    2017-12-01

    Continuous-flow methods introduced by Hayes (Matthews and Hayes, 1978; Freeman et al., 1990; Hayes et al., 1990) for compound-specific isotope analyses (CSIA) transformed how we study the origins and fates of organic compounds. This analytical revolution launched several decades of research in which researchers connect individual molecular structures to diverse environmental and climate processes affecting their isotopic profiles. Among the first applications, and one of the more dramatic isotopically, was tracing the flow of natural methane into cellular carbon and cellular biochemical constituents. Microbial oxidation of methane can be tracked by strongly 13C-depleted organic carbon in early Earth sedimentary environments, in marine and lake-derived biomarkers in oils, and in modern organisms and their environments. These signatures constrain microbial carbon cycling and inform our understanding of ocean redox. The measurement of molecular isotopes has jumped forward once again, and it is now possible to determine isotope abundances at specific positions within increasingly complex organic structures. In addition, recent analytical developments have lowered sample sensitivity limits of CSIA to picomole levels. These new tools have opened new ways to measure methane carbon in the natural environment and within biochemical pathways. This talk will highlight how molecular isotope methods enable us to follow the fate of methane carbon in complex environments and along diverse metabolic pathways, from trace fluids to specific carbon positions within microbial biomarkers.

  13. SCAR Mediates Light-Induced Root Elongation in Arabidopsis through Photoreceptors and Proteasomes[W][OA

    PubMed Central

    Dyachok, Julia; Zhu, Ling; Liao, Fuqi; He, Ji; Huq, Enamul; Blancaflor, Elison B.

    2011-01-01

    The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex. PMID:21972261

  14. Linear analysis near a steady-state of biochemical networks: control analysis, correlation metrics and circuit theory.

    PubMed

    Heuett, William J; Beard, Daniel A; Qian, Hong

    2008-05-15

    Several approaches, including metabolic control analysis (MCA), flux balance analysis (FBA), correlation metric construction (CMC), and biochemical circuit theory (BCT), have been developed for the quantitative analysis of complex biochemical networks. Here, we present a comprehensive theory of linear analysis for nonequilibrium steady-state (NESS) biochemical reaction networks that unites these disparate approaches in a common mathematical framework and thermodynamic basis. In this theory a number of relationships between key matrices are introduced: the matrix A obtained in the standard, linear-dynamic-stability analysis of the steady-state can be decomposed as A = SRT where R and S are directly related to the elasticity-coefficient matrix for the fluxes and chemical potentials in MCA, respectively; the control-coefficients for the fluxes and chemical potentials can be written in terms of RTBS and STBS respectively where matrix B is the inverse of A; the matrix S is precisely the stoichiometric matrix in FBA; and the matrix eAt plays a central role in CMC. One key finding that emerges from this analysis is that the well-known summation theorems in MCA take different forms depending on whether metabolic steady-state is maintained by flux injection or concentration clamping. We demonstrate that if rate-limiting steps exist in a biochemical pathway, they are the steps with smallest biochemical conductances and largest flux control-coefficients. We hypothesize that biochemical networks for cellular signaling have a different strategy for minimizing energy waste and being efficient than do biochemical networks for biosynthesis. We also discuss the intimate relationship between MCA and biochemical systems analysis (BSA).

  15. Ryan Davis | NREL

    Science.gov Websites

    design TEA LCA Biochemical conversion process pathways Algal biomass production and conversion pathways Production," Green Chemistry (2015) Process Design and Economics for the Conversion of Lignocellulosic Production," Applied Energy (2011) Process Design and Economics for Biochemical Conversion of

  16. A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop.

    PubMed

    Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

    2016-06-14

    Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output.

  17. Pathogen ‘Roid Rage: Cholesterol Utilization by Mycobacterium tuberculosis

    PubMed Central

    Wipperman, Matthew F.; Sampson, Nicole S.; Thomas, Suzanne, T.

    2014-01-01

    The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism, and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism. PMID:24611808

  18. Biosynthesis and Metabolic Engineering of Anthocyanins in Arabidopsis thaliana

    PubMed Central

    Shi, Ming-Zhu; Xie, De-Yu

    2014-01-01

    Arabidopsis thaliana is the first model plant, the genome of which has been sequenced. In general, intensive studies on this model plant over the past nearly 30 years have led to many new revolutionary understandings in every single aspect of plant biology. Here, we review the current understanding of anthocyanin biosynthesis in this model plant. Although the investigation of anthocyanin structures in this model plant was not performed until 2002, numerous studies over the past three decades have been conducted to understand the biosynthesis of anthocyanins. To date, it appears that all pathway genes of anthocyanins have been molecularly, genetically and biochemically characterized in this plant. These fundamental accomplishments have made Arabidopsis an ideal model to understand the regulatory mechanisms of anthocyanin pathway. Several studies have revealed that the biosynthesis of anthocyanins is controlled by WD40-bHLH-MYB (WBM) transcription factor complexes under lighting conditions. However, how different regulatory complexes coordinately and specifically regulate the pathway genes of anthocyanins remains unclear. In this review, we discuss current progresses and findings including structural diversity, regulatory properties and metabolic engineering of anthocyanins in Arabidopsis thaliana. PMID:24354533

  19. Synergizing 13C Metabolic Flux Analysis and Metabolic Engineering for Biochemical Production.

    PubMed

    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang

    Metabolic engineering of industrial microorganisms to produce chemicals, fuels, and drugs has attracted increasing interest as it provides an environment-friendly and renewable route that does not depend on depleting petroleum sources. However, the microbial metabolism is so complex that metabolic engineering efforts often have difficulty in achieving a satisfactory yield, titer, or productivity of the target chemical. To overcome this challenge, 13 C Metabolic Flux Analysis ( 13 C-MFA) has been developed to investigate rigorously the cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, 13 C-MFA has been widely used in academic labs and the biotechnology industry to pinpoint the key issues related to microbial-based chemical production and to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this chapter we introduce the basics of 13 C-MFA and illustrate how 13 C-MFA has been applied to synergize with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production.

  20. Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus.

    PubMed

    Boughanemi, Souhela; Lyonnet, Jordan; Infossi, Pascale; Bauzan, Marielle; Kosta, Artémis; Lignon, Sabrina; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2016-08-01

    The Hdr (heterodisulfide reductase)-like enzyme is predicted, from gene transcript profiling experiments previously published, to be essential in oxidative sulfur metabolism in a number of bacteria and archaea. Nevertheless, no biochemical and physicochemical data are available so far about this enzyme. Genes coding for it were identified in Aquifex aeolicus, a Gram-negative, hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium that uses inorganic sulfur compounds as electron donor to grow. We provide biochemical evidence that this Hdr-like enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or elemental sulfur). We demonstrate, by immunolocalization and cell fractionation, that Hdr-like enzyme is associated, presumably monotopically, with the membrane fraction. We show by co-immunoprecipitation assay or partial purification, that the Hdr proteins form a stable complex composed of at least five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present in two forms of high molecular mass on native gel (∼240 and 450 kDa). These studies allow us to propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Reconstructing biochemical pathways from time course data.

    PubMed

    Srividhya, Jeyaraman; Crampin, Edmund J; McSharry, Patrick E; Schnell, Santiago

    2007-03-01

    Time series data on biochemical reactions reveal transient behavior, away from chemical equilibrium, and contain information on the dynamic interactions among reacting components. However, this information can be difficult to extract using conventional analysis techniques. We present a new method to infer biochemical pathway mechanisms from time course data using a global nonlinear modeling technique to identify the elementary reaction steps which constitute the pathway. The method involves the generation of a complete dictionary of polynomial basis functions based on the law of mass action. Using these basis functions, there are two approaches to model construction, namely the general to specific and the specific to general approach. We demonstrate that our new methodology reconstructs the chemical reaction steps and connectivity of the glycolytic pathway of Lactococcus lactis from time course experimental data.

  2. Suppression of fat deposition in broiler chickens by (-)-hydroxycitric acid supplementation: A proteomics perspective

    PubMed Central

    Peng, Mengling; Han, Jing; Li, Longlong; Ma, Haitian

    2016-01-01

    (-)-Hydroxycitric acid (HCA) suppresses fatty acid synthesis in animals, but its biochemical mechanism in poultry is unclear. This study identified the key proteins associated with fat metabolism and elucidated the biochemical mechanism of (-)-HCA in broiler chickens. Four groups (n = 30 each) received a diet supplemented with 0, 1000, 2000 or 3000 mg/kg (-)-HCA for 4 weeks. Of the differentially expressed liver proteins, 40 and 26 were identified in the mitochondrial and cytoplasm respectively. Pyruvate dehydrogenase E1 components (PDHA1 and PDHB), dihydrolipoyl dehydrogenase (DLD), aconitase (ACO2), a-ketoglutarate dehydrogenase complex (DLST), enoyl-CoA hydratase (ECHS1) and phosphoglycerate kinase (PGK) were upregulated, while NADP-dependent malic enzyme (ME1) was downregulated. Biological network analysis showed that the identified proteins were involved in glycometabolism and lipid metabolism, whereas PDHA1, PDHB, ECHS1, and ME1 were identified in the canonical pathway by Ingenuity Pathway Analysis. The data indicated that (-)-HCA inhibited fatty acid synthesis by reducing the acetyl-CoA supply, via promotion of the tricarboxylic acid cycle (upregulation of PDHA1, PDHB, ACO2, and DLST expression) and inhibition of ME1 expression. Moreover, (-)-HCA promoted fatty acid beta-oxidation by upregulating ECHS1 expression. These results reflect a biochemically relevant mechanism of fat reduction by (-)-HCA in broiler chickens. PMID:27586962

  3. ATLAS of Biochemistry: A Repository of All Possible Biochemical Reactions for Synthetic Biology and Metabolic Engineering Studies.

    PubMed

    Hadadi, Noushin; Hafner, Jasmin; Shajkofci, Adrian; Zisaki, Aikaterini; Hatzimanikatis, Vassily

    2016-10-21

    Because the complexity of metabolism cannot be intuitively understood or analyzed, computational methods are indispensable for studying biochemistry and deepening our understanding of cellular metabolism to promote new discoveries. We used the computational framework BNICE.ch along with cheminformatic tools to assemble the whole theoretical reactome from the known metabolome through expansion of the known biochemistry presented in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We constructed the ATLAS of Biochemistry, a database of all theoretical biochemical reactions based on known biochemical principles and compounds. ATLAS includes more than 130 000 hypothetical enzymatic reactions that connect two or more KEGG metabolites through novel enzymatic reactions that have never been reported to occur in living organisms. Moreover, ATLAS reactions integrate 42% of KEGG metabolites that are not currently present in any KEGG reaction into one or more novel enzymatic reactions. The generated repository of information is organized in a Web-based database ( http://lcsb-databases.epfl.ch/atlas/ ) that allows the user to search for all possible routes from any substrate compound to any product. The resulting pathways involve known and novel enzymatic steps that may indicate unidentified enzymatic activities and provide potential targets for protein engineering. Our approach of introducing novel biochemistry into pathway design and associated databases will be important for synthetic biology and metabolic engineering.

  4. A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer

    DTIC Science & Technology

    2014-04-01

    the Fanconi Anemia Pathway- Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer PRINCIPAL INVESTIGATOR...GRANT NUMBER 4. TITLE AND SUBTITLE A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Fanconi anemia is the most prevalent inherited BMF syndromes, caused by mutations in

  5. A Graphical User Interface for a Method to Infer Kinetics and Network Architecture (MIKANA)

    PubMed Central

    Mourão, Márcio A.; Srividhya, Jeyaraman; McSharry, Patrick E.; Crampin, Edmund J.; Schnell, Santiago

    2011-01-01

    One of the main challenges in the biomedical sciences is the determination of reaction mechanisms that constitute a biochemical pathway. During the last decades, advances have been made in building complex diagrams showing the static interactions of proteins. The challenge for systems biologists is to build realistic models of the dynamical behavior of reactants, intermediates and products. For this purpose, several methods have been recently proposed to deduce the reaction mechanisms or to estimate the kinetic parameters of the elementary reactions that constitute the pathway. One such method is MIKANA: Method to Infer Kinetics And Network Architecture. MIKANA is a computational method to infer both reaction mechanisms and estimate the kinetic parameters of biochemical pathways from time course data. To make it available to the scientific community, we developed a Graphical User Interface (GUI) for MIKANA. Among other features, the GUI validates and processes an input time course data, displays the inferred reactions, generates the differential equations for the chemical species in the pathway and plots the prediction curves on top of the input time course data. We also added a new feature to MIKANA that allows the user to exclude a priori known reactions from the inferred mechanism. This addition improves the performance of the method. In this article, we illustrate the GUI for MIKANA with three examples: an irreversible Michaelis–Menten reaction mechanism; the interaction map of chemical species of the muscle glycolytic pathway; and the glycolytic pathway of Lactococcus lactis. We also describe the code and methods in sufficient detail to allow researchers to further develop the code or reproduce the experiments described. The code for MIKANA is open source, free for academic and non-academic use and is available for download (Information S1). PMID:22096591

  6. A graphical user interface for a method to infer kinetics and network architecture (MIKANA).

    PubMed

    Mourão, Márcio A; Srividhya, Jeyaraman; McSharry, Patrick E; Crampin, Edmund J; Schnell, Santiago

    2011-01-01

    One of the main challenges in the biomedical sciences is the determination of reaction mechanisms that constitute a biochemical pathway. During the last decades, advances have been made in building complex diagrams showing the static interactions of proteins. The challenge for systems biologists is to build realistic models of the dynamical behavior of reactants, intermediates and products. For this purpose, several methods have been recently proposed to deduce the reaction mechanisms or to estimate the kinetic parameters of the elementary reactions that constitute the pathway. One such method is MIKANA: Method to Infer Kinetics And Network Architecture. MIKANA is a computational method to infer both reaction mechanisms and estimate the kinetic parameters of biochemical pathways from time course data. To make it available to the scientific community, we developed a Graphical User Interface (GUI) for MIKANA. Among other features, the GUI validates and processes an input time course data, displays the inferred reactions, generates the differential equations for the chemical species in the pathway and plots the prediction curves on top of the input time course data. We also added a new feature to MIKANA that allows the user to exclude a priori known reactions from the inferred mechanism. This addition improves the performance of the method. In this article, we illustrate the GUI for MIKANA with three examples: an irreversible Michaelis-Menten reaction mechanism; the interaction map of chemical species of the muscle glycolytic pathway; and the glycolytic pathway of Lactococcus lactis. We also describe the code and methods in sufficient detail to allow researchers to further develop the code or reproduce the experiments described. The code for MIKANA is open source, free for academic and non-academic use and is available for download (Information S1).

  7. Modulation of inflammation and disease tolerance by DNA damage response pathways.

    PubMed

    Neves-Costa, Ana; Moita, Luis F

    2017-03-01

    The accurate replication and repair of DNA is central to organismal survival. This process is challenged by the many factors that can change genetic information such as replication errors and direct damage to the DNA molecule by chemical and physical agents. DNA damage can also result from microorganism invasion as an integral step of their life cycle or as collateral damage from host defense mechanisms against pathogens. Here we review the complex crosstalk of DNA damage response and immune response pathways that might be evolutionarily connected and argue that DNA damage response pathways can be explored therapeutically to induce disease tolerance through the activation of tissue damage control processes. Such approach may constitute the missing pillar in the treatment of critical illnesses caused by multiple organ failure, such as sepsis and septic shock. © 2016 Federation of European Biochemical Societies.

  8. Construction and engineering of large biochemical pathways via DNA assembler

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2015-01-01

    Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

  9. Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short-chain fatty acids.

    PubMed

    Hackmann, Timothy J; Ngugi, David Kamanda; Firkins, Jeffrey L; Tao, Junyi

    2017-11-01

    Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here, we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% of bacteria had an atypical pathway for propionate or succinate formation; 6% of bacteria had an atypical pathway for butyrate formation and 33% of bacteria had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways - a common goal of omics studies - could be incorrect if well-recognized pathways are used for reference. Furthermore, it calls for renewed efforts to delineate fermentation pathways biochemically. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. Calculation of biochemical net reactions and pathways by using matrix operations.

    PubMed Central

    Alberty, R A

    1996-01-01

    Pathways for net biochemical reactions can be calculated by using a computer program that solves systems of linear equations. The coefficients in the linear equations are the stoichiometric numbers in the biochemical equations for the system. The solution of the system of linear equations is a vector of the stoichiometric numbers of the reactions in the pathway for the net reaction; this is referred to as the pathway vector. The pathway vector gives the number of times the various reactions have to occur to produce the desired net reaction. Net reactions may involve unknown numbers of ATP, ADP, and Pi molecules. The numbers of ATP, ADP, and Pi in a desired net reaction can be calculated in a two-step process. In the first step, the pathway is calculated by solving the system of linear equations for an abbreviated stoichiometric number matrix without ATP, ADP, Pi, NADred, and NADox. In the second step, the stoichiometric numbers in the desired net reaction, which includes ATP, ADP, Pi, NADred, and NADox, are obtained by multiplying the full stoichiometric number matrix by the calculated pathway vector. PMID:8804633

  11. Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315*

    PubMed Central

    Law, Adrienne; Boulanger, Martin J.

    2011-01-01

    The phenylacetic acid (PAA) degradation pathway is the sole aerobic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaK1 and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct conformations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this prediction, with PaaK1 possessing a lower Km for phenylacetic acid and better able to accommodate 3′ and 4′ substitutions on the phenyl ring. Collectively, these results offer detailed insight into the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia. PMID:21388965

  12. Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree

    PubMed Central

    Rangiah, Kannan; Mahesh, HB; Rajamani, Anantharamanan; Shirke, Meghana D.; Russiachand, Heikham; Loganathan, Ramya Malarini; Shankara Lingu, Chandana; Siddappa, Shilpa; Ramamurthy, Aishwarya; Sathyanarayana, BN

    2015-01-01

    Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC–600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways. PMID:26290780

  13. Detecting breakdown points in metabolic networks.

    PubMed

    Tagore, Somnath; De, Rajat K

    2011-12-14

    A complex network of biochemical reactions present in an organism generates various biological moieties necessary for its survival. It is seen that biological systems are robust to genetic and environmental changes at all levels of organization. Functions of various organisms are sustained against mutational changes by using alternative pathways. It is also seen that if any one of the paths for production of the same metabolite is hampered, an alternate path tries to overcome this defect and helps in combating the damage. Certain physical, chemical or genetic change in any of the precursor substrate of a biochemical reaction may damage the production of the ultimate product. We employ a quantitative approach for simulating this phenomena of causing a physical change in the biochemical reactions by performing external perturbations to 12 metabolic pathways under carbohydrate metabolism in Saccharomyces cerevisae as well as 14 metabolic pathways under carbohydrate metabolism in Homo sapiens. Here, we investigate the relationship between structure and degree of compatibility of metabolites against external perturbations, i.e., robustness. Robustness can also be further used to identify the extent to which a metabolic pathway can resist a mutation event. Biological networks with a certain connectivity distribution may be very resilient to a particular attack but not to another. The goal of this work is to determine the exact boundary of network breakdown due to both random and targeted attack, thereby analyzing its robustness. We also find that compared to various non-standard models, metabolic networks are exceptionally robust. Here, we report the use of a 'Resilience-based' score for enumerating the concept of 'network-breakdown'. We also use this approach for analyzing metabolite essentiality providing insight into cellular robustness that can be further used for future drug development. We have investigated the behavior of metabolic pathways under carbohydrate metabolism in S. cerevisae and H. sapiens against random and targeted attack. Both random as well as targeted resilience were calculated by formulating a measure, that we termed as 'Resilience score'. Datasets of metabolites were collected for 12 metabolic pathways belonging to carbohydrate metabolism in S. cerevisae and 14 metabolic pathways belonging to carbohydrate metabolism in H. sapiens from Kyoto Encyclopedia for Genes and Genomes (KEGG). Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. A Method for Finding Metabolic Pathways Using Atomic Group Tracking.

    PubMed

    Huang, Yiran; Zhong, Cheng; Lin, Hai Xiang; Wang, Jianyi

    2017-01-01

    A fundamental computational problem in metabolic engineering is to find pathways between compounds. Pathfinding methods using atom tracking have been widely used to find biochemically relevant pathways. However, these methods require the user to define the atoms to be tracked. This may lead to failing to predict the pathways that do not conserve the user-defined atoms. In this work, we propose a pathfinding method called AGPathFinder to find biochemically relevant metabolic pathways between two given compounds. In AGPathFinder, we find alternative pathways by tracking the movement of atomic groups through metabolic networks and use combined information of reaction thermodynamics and compound similarity to guide the search towards more feasible pathways and better performance. The experimental results show that atomic group tracking enables our method to find pathways without the need of defining the atoms to be tracked, avoid hub metabolites, and obtain biochemically meaningful pathways. Our results also demonstrate that atomic group tracking, when incorporated with combined information of reaction thermodynamics and compound similarity, improves the quality of the found pathways. In most cases, the average compound inclusion accuracy and reaction inclusion accuracy for the top resulting pathways of our method are around 0.90 and 0.70, respectively, which are better than those of the existing methods. Additionally, AGPathFinder provides the information of thermodynamic feasibility and compound similarity for the resulting pathways.

  15. A Method for Finding Metabolic Pathways Using Atomic Group Tracking

    PubMed Central

    Zhong, Cheng; Lin, Hai Xiang; Wang, Jianyi

    2017-01-01

    A fundamental computational problem in metabolic engineering is to find pathways between compounds. Pathfinding methods using atom tracking have been widely used to find biochemically relevant pathways. However, these methods require the user to define the atoms to be tracked. This may lead to failing to predict the pathways that do not conserve the user-defined atoms. In this work, we propose a pathfinding method called AGPathFinder to find biochemically relevant metabolic pathways between two given compounds. In AGPathFinder, we find alternative pathways by tracking the movement of atomic groups through metabolic networks and use combined information of reaction thermodynamics and compound similarity to guide the search towards more feasible pathways and better performance. The experimental results show that atomic group tracking enables our method to find pathways without the need of defining the atoms to be tracked, avoid hub metabolites, and obtain biochemically meaningful pathways. Our results also demonstrate that atomic group tracking, when incorporated with combined information of reaction thermodynamics and compound similarity, improves the quality of the found pathways. In most cases, the average compound inclusion accuracy and reaction inclusion accuracy for the top resulting pathways of our method are around 0.90 and 0.70, respectively, which are better than those of the existing methods. Additionally, AGPathFinder provides the information of thermodynamic feasibility and compound similarity for the resulting pathways. PMID:28068354

  16. ReactPRED: a tool to predict and analyze biochemical reactions.

    PubMed

    Sivakumar, Tadi Venkata; Giri, Varun; Park, Jin Hwan; Kim, Tae Yong; Bhaduri, Anirban

    2016-11-15

    Biochemical pathways engineering is often used to synthesize or degrade target chemicals. In silico screening of the biochemical transformation space allows predicting feasible reactions, constituting these pathways. Current enabling tools are customized to predict reactions based on pre-defined biochemical transformations or reaction rule sets. Reaction rule sets are usually curated manually and tailored to specific applications. They are not exhaustive. In addition, current systems are incapable of regulating and refining data with an aim to tune specificity and sensitivity. A robust and flexible tool that allows automated reaction rule set creation along with regulated pathway prediction and analyses is a need. ReactPRED aims to address the same. ReactPRED is an open source flexible and customizable tool enabling users to predict biochemical reactions and pathways. The tool allows automated reaction rule creation from a user defined reaction set. Additionally, reaction rule degree and rule tolerance features allow refinement of predicted data. It is available as a flexible graphical user interface and a console application. ReactPRED is available at: https://sourceforge.net/projects/reactpred/ CONTACT: anirban.b@samsung.com or ty76.kim@samsung.comSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Linear analysis near a steady-state of biochemical networks: Control analysis, correlation metrics and circuit theory

    PubMed Central

    Heuett, William J; Beard, Daniel A; Qian, Hong

    2008-01-01

    Background Several approaches, including metabolic control analysis (MCA), flux balance analysis (FBA), correlation metric construction (CMC), and biochemical circuit theory (BCT), have been developed for the quantitative analysis of complex biochemical networks. Here, we present a comprehensive theory of linear analysis for nonequilibrium steady-state (NESS) biochemical reaction networks that unites these disparate approaches in a common mathematical framework and thermodynamic basis. Results In this theory a number of relationships between key matrices are introduced: the matrix A obtained in the standard, linear-dynamic-stability analysis of the steady-state can be decomposed as A = SRT where R and S are directly related to the elasticity-coefficient matrix for the fluxes and chemical potentials in MCA, respectively; the control-coefficients for the fluxes and chemical potentials can be written in terms of RTBS and STBS respectively where matrix B is the inverse of A; the matrix S is precisely the stoichiometric matrix in FBA; and the matrix eAt plays a central role in CMC. Conclusion One key finding that emerges from this analysis is that the well-known summation theorems in MCA take different forms depending on whether metabolic steady-state is maintained by flux injection or concentration clamping. We demonstrate that if rate-limiting steps exist in a biochemical pathway, they are the steps with smallest biochemical conductances and largest flux control-coefficients. We hypothesize that biochemical networks for cellular signaling have a different strategy for minimizing energy waste and being efficient than do biochemical networks for biosynthesis. We also discuss the intimate relationship between MCA and biochemical systems analysis (BSA). PMID:18482450

  18. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

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

    Wubben, T.; Mesecar, A.D.; UIC)

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observedmore » in the MtPPAT-CoA complex.« less

  19. Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

    PubMed

    Wieczorek, Andrew S; Martin, Vincent J J

    2012-12-15

    The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

  20. Exercise-induced biochemical changes and their potential influence on cancer: a scientific review

    PubMed Central

    Thomas, Robert James; Kenfield, Stacey A; Jimenez, Alfonso

    2017-01-01

    Aim To review and discuss the available international literature regarding the indirect and direct biochemical mechanisms that occur after exercise, which could positively, or negatively, influence oncogenic pathways. Methods The PubMed, MEDLINE, Embase and Cochrane libraries were searched for papers up to July 2016 addressing biochemical changes after exercise with a particular reference to cancer. The three authors independently assessed their appropriateness for inclusion in this review based on their scientific quality and relevance. Results 168 papers were selected and categorised into indirect and direct biochemical pathways. The indirect effects included changes in vitamin D, weight reduction, sunlight exposure and improved mood. The direct effects included insulin-like growth factor, epigenetic effects on gene expression and DNA repair, vasoactive intestinal peptide, oxidative stress and antioxidant pathways, heat shock proteins, testosterone, irisin, immunity, chronic inflammation and prostaglandins, energy metabolism and insulin resistance. Summary Exercise is one of several lifestyle factors known to lower the risk of developing cancer and is associated with lower relapse rates and better survival. This review highlights the numerous biochemical processes, which explain these potential anticancer benefits. PMID:27993842

  1. Poliovirus Cell Entry: Common Structural Themes in Viral Cell Entry Pathways

    PubMed Central

    Hogle, James M.

    2006-01-01

    Structural studies of polio- and closely related viruses have provided a series of snapshots along their cell entry pathways. Based on the structures and related kinetic, biochemical, and genetic studies, we have proposed a model for the cell entry pathway for polio- and closely related viruses. In this model a maturation cleavage of a capsid protein precursor locks the virus in a metastable state, and the receptor acts like a transition-state catalyst to overcome an energy barrier and release the mature virion from the metastable state. This initiates a series of conformational changes that allow the virus to attach to membranes, form a pore, and finally release its RNA genome into the cytoplasm. This model has striking parallels with emerging models for the maturation and cell entry of more complex enveloped viruses such as influenza virus and HIV. PMID:12142481

  2. In vitro DNA SCRaMbLE.

    PubMed

    Wu, Yi; Zhu, Rui-Ying; Mitchell, Leslie A; Ma, Lu; Liu, Rui; Zhao, Meng; Jia, Bin; Xu, Hui; Li, Yun-Xiang; Yang, Zu-Ming; Ma, Yuan; Li, Xia; Liu, Hong; Liu, Duo; Xiao, Wen-Hai; Zhou, Xiao; Li, Bing-Zhi; Yuan, Ying-Jin; Boeke, Jef D

    2018-05-22

    The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

  3. Ecm33 is a novel factor involved in efficient glucose uptake for nutrition-responsive TORC1 signaling in yeast.

    PubMed

    Umekawa, Midori; Ujihara, Masato; Nakai, Daiki; Takematsu, Hiromu; Wakayama, Mamoru

    2017-11-01

    Glucose uptake is crucial for providing both an energy source and a signal that regulates cell proliferation. Therefore, it is important to clarify the mechanisms underlying glucose uptake and its transmission to intracellular signaling pathways. In this study, we searched for a novel regulatory factor involved in glucose-induced signaling by using Saccharomyces cerevisiae as a eukaryotic model. Requirement of the extracellular protein Ecm33 in efficient glucose uptake and full activation of the nutrient-responsive TOR kinase complex 1 (TORC1) signaling pathway is shown. Cells lacking Ecm33 elicit a series of starvation-induced pathways even in the presence of extracellular high glucose concentration. This results in delayed cell proliferation, reduced ATP, induction of autophagy, and dephosphorylation of the TORC1 substrates Atg13 and Sch9. © 2017 Federation of European Biochemical Societies.

  4. Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

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

    Barajas, Jesus F.; Zargar, Amin; Pang, Bo

    Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

  5. Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

    DOE PAGES

    Barajas, Jesus F.; Zargar, Amin; Pang, Bo; ...

    2018-03-30

    Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

  6. Physical understanding of complex multiscale biochemical models via algorithmic simplification: Glycolysis in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Kourdis, Panayotis D.; Steuer, Ralf; Goussis, Dimitris A.

    2010-09-01

    Large-scale models of cellular reaction networks are usually highly complex and characterized by a wide spectrum of time scales, making a direct interpretation and understanding of the relevant mechanisms almost impossible. We address this issue by demonstrating the benefits provided by model reduction techniques. We employ the Computational Singular Perturbation (CSP) algorithm to analyze the glycolytic pathway of intact yeast cells in the oscillatory regime. As a primary object of research for many decades, glycolytic oscillations represent a paradigmatic candidate for studying biochemical function and mechanisms. Using a previously published full-scale model of glycolysis, we show that, due to fast dissipative time scales, the solution is asymptotically attracted on a low dimensional manifold. Without any further input from the investigator, CSP clarifies several long-standing questions in the analysis of glycolytic oscillations, such as the origin of the oscillations in the upper part of glycolysis, the importance of energy and redox status, as well as the fact that neither the oscillations nor cell-cell synchronization can be understood in terms of glycolysis as a simple linear chain of sequentially coupled reactions.

  7. Solid-Phase Enrichment and Analysis of Azide-Labeled Natural Products: Fishing Downstream of Biochemical Pathways.

    PubMed

    Pérez, Alexander J; Wesche, Frank; Adihou, Hélène; Bode, Helge B

    2016-01-11

    Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development. For bacterial secondary metabolism, a far broader tolerance for "clickable" precursors was observed than in ribosomal proteinogenesis, thus making this method a surprisingly valuable tool for the tracking and discovery of compounds within the cellular biochemical network. The implementation of this method has led to the identification of several new compounds from the bacterial genera Photorhabdus and Xenorhabdus, clearly proving its power. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Reactions of vitamin A with acceptors of electrons. Interactions with iodine and the formation of iodide

    PubMed Central

    Lucy, J. A.; Lichti, F. Ulrike

    1969-01-01

    1. The reactions of retinol and retinoic acid with iodine were investigated since knowledge of the chemical reactions of vitamin A with acceptors of electrons may shed light on its biochemical mode of action. 2. Colloidal retinol, but not retinoic acid, reacts with iodine to yield a blue–green complex that rapidly decomposes, giving iodide and an unknown species with λmax. at 870mμ. 3. In addition, both retinol and retinoic acid reduce iodine to iodide by a reaction that does not involve an intermediate coloured complex; this reaction appears to yield unstable carbonium ion derivatives of the vitamin. 4. The presence of water greatly facilitates the production of iodide from vitamin A and iodine. 5. Possible chemical pathways involved in these reactions are discussed. 6. It is suggested that the chemical properties of retinol and retinoic acid that underlie their biochemical behaviour might be apparent only when the molecules are at a lipid–water interface, and that vitamin A might be expected to react with a number of different electron acceptors in vivo. PMID:5801297

  9. Quantitative trait loci and metabolic pathways

    PubMed Central

    McMullen, M. D.; Byrne, P. F.; Snook, M. E.; Wiseman, B. R.; Lee, E. A.; Widstrom, N. W.; Coe, E. H.

    1998-01-01

    The interpretation of quantitative trait locus (QTL) studies is limited by the lack of information on metabolic pathways leading to most economic traits. Inferences about the roles of the underlying genes with a pathway or the nature of their interaction with other loci are generally not possible. An exception is resistance to the corn earworm Helicoverpa zea (Boddie) in maize (Zea mays L.) because of maysin, a C-glycosyl flavone synthesized in silks via a branch of the well characterized flavonoid pathway. Our results using flavone synthesis as a model QTL system indicate: (i) the importance of regulatory loci as QTLs, (ii) the importance of interconnecting biochemical pathways on product levels, (iii) evidence for “channeling” of intermediates, allowing independent synthesis of related compounds, (iv) the utility of QTL analysis in clarifying the role of specific genes in a biochemical pathway, and (v) identification of a previously unknown locus on chromosome 9S affecting flavone level. A greater understanding of the genetic basis of maysin synthesis and associated corn earworm resistance should lead to improved breeding strategies. More broadly, the insights gained in relating a defined genetic and biochemical pathway affecting a quantitative trait should enhance interpretation of the biological basis of variation for other quantitative traits. PMID:9482823

  10. A bacterial hydrogen-dependent CO2 reductase forms filamentous structures.

    PubMed

    Schuchmann, Kai; Vonck, Janet; Müller, Volker

    2016-04-01

    Interconversion of CO2 and formic acid is an important reaction in bacteria. A novel enzyme complex that directly utilizes molecular hydrogen as electron donor for the reversible reduction of CO2 has recently been identified in the Wood-Ljungdahl pathway of an acetogenic bacterium. This pathway is utilized for carbon fixation as well as energy conservation. Here we describe the further characterization of the quaternary structure of this enzyme complex and the unexpected behavior of this enzyme in polymerizing into filamentous structures. Polymerization of metabolic enzymes into similar structures has been observed only in rare cases but the increasing number of examples point towards a more general characteristic of enzyme functioning. Polymerization of the purified enzyme into ordered filaments of more than 0.1 μm in length was only dependent on the presence of divalent cations. Polymerization was a reversible process and connected to the enzymatic activity of the oxygen-sensitive enzyme with the filamentous form being the most active state. © 2016 Federation of European Biochemical Societies.

  11. Fast and Precise Emulation of Stochastic Biochemical Reaction Networks With Amplified Thermal Noise in Silicon Chips.

    PubMed

    Kim, Jaewook; Woo, Sung Sik; Sarpeshkar, Rahul

    2018-04-01

    The analysis and simulation of complex interacting biochemical reaction pathways in cells is important in all of systems biology and medicine. Yet, the dynamics of even a modest number of noisy or stochastic coupled biochemical reactions is extremely time consuming to simulate. In large part, this is because of the expensive cost of random number and Poisson process generation and the presence of stiff, coupled, nonlinear differential equations. Here, we demonstrate that we can amplify inherent thermal noise in chips to emulate randomness physically, thus alleviating these costs significantly. Concurrently, molecular flux in thermodynamic biochemical reactions maps to thermodynamic electronic current in a transistor such that stiff nonlinear biochemical differential equations are emulated exactly in compact, digitally programmable, highly parallel analog "cytomorphic" transistor circuits. For even small-scale systems involving just 80 stochastic reactions, our 0.35-μm BiCMOS chips yield a 311× speedup in the simulation time of Gillespie's stochastic algorithm over COPASI, a fast biochemical-reaction software simulator that is widely used in computational biology; they yield a 15 500× speedup over equivalent MATLAB stochastic simulations. The chip emulation results are consistent with these software simulations over a large range of signal-to-noise ratios. Most importantly, our physical emulation of Poisson chemical dynamics does not involve any inherently sequential processes and updates such that, unlike prior exact simulation approaches, they are parallelizable, asynchronous, and enable even more speedup for larger-size networks.

  12. A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop

    PubMed Central

    Fuentes, Paulina; Zhou, Fei; Erban, Alexander; Karcher, Daniel; Kopka, Joachim; Bock, Ralph

    2016-01-01

    Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output. DOI: http://dx.doi.org/10.7554/eLife.13664.001 PMID:27296645

  13. Multi-membership gene regulation in pathway based microarray analysis

    PubMed Central

    2011-01-01

    Background Gene expression analysis has been intensively researched for more than a decade. Recently, there has been elevated interest in the integration of microarray data analysis with other types of biological knowledge in a holistic analytical approach. We propose a methodology that can be facilitated for pathway based microarray data analysis, based on the observation that a substantial proportion of genes present in biochemical pathway databases are members of a number of distinct pathways. Our methodology aims towards establishing the state of individual pathways, by identifying those truly affected by the experimental conditions based on the behaviour of such genes. For that purpose it considers all the pathways in which a gene participates and the general census of gene expression per pathway. Results We utilise hill climbing, simulated annealing and a genetic algorithm to analyse the consistency of the produced results, through the application of fuzzy adjusted rand indexes and hamming distance. All algorithms produce highly consistent genes to pathways allocations, revealing the contribution of genes to pathway functionality, in agreement with current pathway state visualisation techniques, with the simulated annealing search proving slightly superior in terms of efficiency. Conclusions We show that the expression values of genes, which are members of a number of biochemical pathways or modules, are the net effect of the contribution of each gene to these biochemical processes. We show that by manipulating the pathway and module contribution of such genes to follow underlying trends we can interpret microarray results centred on the behaviour of these genes. PMID:21939531

  14. Multi-membership gene regulation in pathway based microarray analysis.

    PubMed

    Pavlidis, Stelios P; Payne, Annette M; Swift, Stephen M

    2011-09-22

    Gene expression analysis has been intensively researched for more than a decade. Recently, there has been elevated interest in the integration of microarray data analysis with other types of biological knowledge in a holistic analytical approach. We propose a methodology that can be facilitated for pathway based microarray data analysis, based on the observation that a substantial proportion of genes present in biochemical pathway databases are members of a number of distinct pathways. Our methodology aims towards establishing the state of individual pathways, by identifying those truly affected by the experimental conditions based on the behaviour of such genes. For that purpose it considers all the pathways in which a gene participates and the general census of gene expression per pathway. We utilise hill climbing, simulated annealing and a genetic algorithm to analyse the consistency of the produced results, through the application of fuzzy adjusted rand indexes and hamming distance. All algorithms produce highly consistent genes to pathways allocations, revealing the contribution of genes to pathway functionality, in agreement with current pathway state visualisation techniques, with the simulated annealing search proving slightly superior in terms of efficiency. We show that the expression values of genes, which are members of a number of biochemical pathways or modules, are the net effect of the contribution of each gene to these biochemical processes. We show that by manipulating the pathway and module contribution of such genes to follow underlying trends we can interpret microarray results centred on the behaviour of these genes.

  15. In search of cellular control: signal transduction in context

    NASA Technical Reports Server (NTRS)

    Ingber, D.

    1998-01-01

    The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

  16. Truncated somatostatin receptor 5 may modulate therapy response to somatostatin analogues--Observations in two patients with acromegaly and severe headache.

    PubMed

    Marina, Djordje; Burman, Pia; Klose, Marianne; Casar-Borota, Olivera; Luque, Raúl M; Castaño, Justo P; Feldt-Rasmussen, Ulla

    2015-10-01

    Somatotropinomas have unique "fingerprints" of somatostatin receptor (sst) expression, which are targets in treatment of acromegaly with somatostatin analogues (SSAs). However, a significant expression of sst is not always related to the biochemical response to SSAs. Headache is a common complaint in acromegaly and considered a clinical marker of disease activity. SSAs are reported to have an own analgesic effect, but the sst involved are unknown. We investigated sst expression in two acromegalic patients with severe headache and no biochemical effects of octreotide, but a good response to pasireotide. We searched the literature for determinants of biochemical and analgesic effects of SSAs in somatotropinomas. Case 1 had no biochemical or analgesic effects of octreotide, a semi-selective SSA, but a rapid and significant effect of pasireotide, a pan-SSA. Case 2 demonstrated discordance between analgesic and biochemical effects of octreotide, in that headache disappeared, but without biochemical improvement. In contrast, pasireotide normalized insulin-like growth factor 1. Both adenomas were sparsely granulated and had strong membranous expressions of sst2a in 50-75% and sst5 in 75-100% of tumor cells. The truncated sst5 variant TMD4 (sst5TMD4) showed expression in 20-57% of tumor cells. A poor biochemical response to octreotide may be associated with tumor expression of a truncated sst5 variant, despite abundant sst2a expression, suggesting an influence from variant sst5 on common sst signaling pathways. Furthermore, unrelated analgesic and biochemical effects of SSAs supported a complex pathogenesis of acromegaly-associated headache. Finally, assessment of truncated sst5 in addition to full length sst could be important for a choice of postoperative SSA treatment in somatotropinomas. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation

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

    Peng, Qianqian; Lan, Xi; Wang, Chen

    Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together,more » these data reveal a new mechanism for immune evasion of PKV. - Highlights: •PKV VP3 inhibits the IFN-β-triggered signaling pathway. •VP3 associates with STAT2 and IRF9. •VP3 blocks the STAT2-IRF9 nuclear translocation. •VP3 utilizes a novel strategy for innate immune evasion.« less

  18. Identifying metabolic pathways for production of extracellular polymeric substances by the diatom Fragilariopsis cylindrus inhabiting sea ice.

    PubMed

    Aslam, Shazia N; Strauss, Jan; Thomas, David N; Mock, Thomas; Underwood, Graham J C

    2018-05-01

    Diatoms are significant primary producers in sea ice, an ephemeral habitat with steep vertical gradients of temperature and salinity characterizing the ice matrix environment. To cope with the variable and challenging conditions, sea ice diatoms produce polysaccharide-rich extracellular polymeric substances (EPS) that play important roles in adhesion, cell protection, ligand binding and as organic carbon sources. Significant differences in EPS concentrations and chemical composition corresponding to temperature and salinity gradients were present in sea ice from the Weddell Sea and Eastern Antarctic regions of the Southern Ocean. To reconstruct the first metabolic pathway for EPS production in diatoms, we exposed Fragilariopsis cylindrus, a key bi-polar diatom species, to simulated sea ice formation. Transcriptome profiling under varying conditions of EPS production identified a significant number of genes and divergent alleles. Their complex differential expression patterns under simulated sea ice formation was aligned with physiological and biochemical properties of the cells, and with field measurements of sea ice EPS characteristics. Thus, the molecular complexity of the EPS pathway suggests metabolic plasticity in F. cylindrus is required to cope with the challenging conditions of the highly variable and extreme sea ice habitat.

  19. Nutritional Lipidomics: Molecular Metabolism, Analytics, and Diagnostics

    PubMed Central

    Smilowitz, Jennifer T.; Zivkovic, Angela M.; Wan, Yu-Jui Yvonne; Watkins, Steve M.; Nording, Malin L.; Hammock, Bruce D.; German, J. Bruce

    2013-01-01

    The field of lipidomics is providing nutritional science a more comprehensive view of lipid intermediates. Lipidomics research takes advantage of the increase in accuracy and sensitivity of mass detection of mass spectrometry with new bioinformatics toolsets to characterize the structures and abundances of complex lipids. Yet, translating lipidomics to practice via nutritional interventions is still in its infancy. No single instrumentation platform is able to solve the varying analytical challenges of the different molecular lipid species. Biochemical pathways of lipid metabolism remain incomplete and the tools to map lipid compositional data to pathways are still being assembled. Biology itself is dauntingly complex and simply separating biological structures remains a key challenge to lipidomics. Nonetheless, the strategy of combining tandem analytical methods to perform the sensitive, high-throughput, quantitative and comprehensive analysis of lipid metabolites of very large numbers of molecules is poised to drive the field forward rapidly. Among the next steps for nutrition to understand the changes in structures, compositions and function of lipid biomolecules in response to diet is to describe their distribution within discrete functional compartments-lipoproteins. Additionally, lipidomics must tackle the task of assigning the functions of lipids as signaling molecules, nutrient sensors, and intermediates of metabolic pathways. PMID:23818328

  20. Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

    PubMed

    Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

    2011-04-01

    Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

  1. Discovery of biochemical biomarkers for aggression: A role for metabolomics in psychiatry.

    PubMed

    Hagenbeek, Fiona A; Kluft, Cornelis; Hankemeier, Thomas; Bartels, Meike; Draisma, Harmen H M; Middeldorp, Christel M; Berger, Ruud; Noto, Antonio; Lussu, Milena; Pool, René; Fanos, Vassilios; Boomsma, Dorret I

    2016-07-01

    Human aggression encompasses a wide range of behaviors and is related to many psychiatric disorders. We introduce the different classification systems of aggression and related disorders as a basis for discussing biochemical biomarkers and then present an overview of studies in humans (published between 1990 and 2015) that reported statistically significant associations of biochemical biomarkers with aggression, DSM-IV disorders involving aggression, and their subtypes. The markers are of different types, including inflammation markers, neurotransmitters, lipoproteins, and hormones from various classes. Most studies focused on only a limited portfolio of biomarkers, frequently a specific class only. When integrating the data, it is clear that compounds from several biological pathways have been found to be associated with aggressive behavior, indicating complexity and the need for a broad approach. In the second part of the paper, using examples from the aggression literature and psychiatric metabolomics studies, we argue that a better understanding of aggression would benefit from a more holistic approach such as provided by metabolomics. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Proline Precursors and Collagen Synthesis: Biochemical Challenges of Nutrient Supplementation and Wound Healing.

    PubMed

    Albaugh, Vance L; Mukherjee, Kaushik; Barbul, Adrian

    2017-11-01

    Wound healing is a complex process marked by highly coordinated immune fluxes into an area of tissue injury; these are required for re-establishment of normal tissue integrity. Along with this cascade of cellular players, wound healing also requires coordinated flux through a number of biochemical pathways, leading to synthesis of collagen and recycling or removal of damaged tissues. The availability of nutrients, especially amino acids, is critical for wound healing, and enteral supplementation has been intensely studied as a potential mechanism to augment wound healing-either by increasing tensile strength, decreasing healing time, or both. From a practical standpoint, although enteral nutrient supplementation may seem like a reasonable strategy to augment healing, a number of biochemical and physiologic barriers exist that limit this strategy. In this critical review, the physiology of enteral amino acid metabolism and supplementation and challenges therein are discussed in the context of splanchnic physiology and biochemistry. Additionally, a review of studies examining various methods of amino acid supplementation and the associated effects on wound outcomes are discussed. © 2017 American Society for Nutrition.

  3. The geometric nature of weights in real complex networks

    NASA Astrophysics Data System (ADS)

    Allard, Antoine; Serrano, M. Ángeles; García-Pérez, Guillermo; Boguñá, Marián

    2017-01-01

    The topology of many real complex networks has been conjectured to be embedded in hidden metric spaces, where distances between nodes encode their likelihood of being connected. Besides of providing a natural geometrical interpretation of their complex topologies, this hypothesis yields the recipe for sustainable Internet's routing protocols, sheds light on the hierarchical organization of biochemical pathways in cells, and allows for a rich characterization of the evolution of international trade. Here we present empirical evidence that this geometric interpretation also applies to the weighted organization of real complex networks. We introduce a very general and versatile model and use it to quantify the level of coupling between their topology, their weights and an underlying metric space. Our model accurately reproduces both their topology and their weights, and our results suggest that the formation of connections and the assignment of their magnitude are ruled by different processes.

  4. Matt Wecker | NREL

    Science.gov Websites

    acetaldehyde from bacteria. The idea was to short-sheet the ethanol fermentation pathway to produce ; Biochem. (1995) "Fermentation strategies: Acetaldehyde or ethanol?," Process Biochem. (1987

  5. Analyzing the genes related to Alzheimer's disease via a network and pathway-based approach.

    PubMed

    Hu, Yan-Shi; Xin, Juncai; Hu, Ying; Zhang, Lei; Wang, Ju

    2017-04-27

    Our understanding of the molecular mechanisms underlying Alzheimer's disease (AD) remains incomplete. Previous studies have revealed that genetic factors provide a significant contribution to the pathogenesis and development of AD. In the past years, numerous genes implicated in this disease have been identified via genetic association studies on candidate genes or at the genome-wide level. However, in many cases, the roles of these genes and their interactions in AD are still unclear. A comprehensive and systematic analysis focusing on the biological function and interactions of these genes in the context of AD will therefore provide valuable insights to understand the molecular features of the disease. In this study, we collected genes potentially associated with AD by screening publications on genetic association studies deposited in PubMed. The major biological themes linked with these genes were then revealed by function and biochemical pathway enrichment analysis, and the relation between the pathways was explored by pathway crosstalk analysis. Furthermore, the network features of these AD-related genes were analyzed in the context of human interactome and an AD-specific network was inferred using the Steiner minimal tree algorithm. We compiled 430 human genes reported to be associated with AD from 823 publications. Biological theme analysis indicated that the biological processes and biochemical pathways related to neurodevelopment, metabolism, cell growth and/or survival, and immunology were enriched in these genes. Pathway crosstalk analysis then revealed that the significantly enriched pathways could be grouped into three interlinked modules-neuronal and metabolic module, cell growth/survival and neuroendocrine pathway module, and immune response-related module-indicating an AD-specific immune-endocrine-neuronal regulatory network. Furthermore, an AD-specific protein network was inferred and novel genes potentially associated with AD were identified. By means of network and pathway-based methodology, we explored the pathogenetic mechanism underlying AD at a systems biology level. Results from our work could provide valuable clues for understanding the molecular mechanism underlying AD. In addition, the framework proposed in this study could be used to investigate the pathological molecular network and genes relevant to other complex diseases or phenotypes.

  6. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication

    PubMed Central

    Gu, Haidong

    2016-01-01

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  7. Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

    PubMed

    Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

    2016-12-27

    The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

  8. Systems biology by the rules: hybrid intelligent systems for pathway modeling and discovery.

    PubMed

    Bosl, William J

    2007-02-15

    Expert knowledge in journal articles is an important source of data for reconstructing biological pathways and creating new hypotheses. An important need for medical research is to integrate this data with high throughput sources to build useful models that span several scales. Researchers traditionally use mental models of pathways to integrate information and development new hypotheses. Unfortunately, the amount of information is often overwhelming and these are inadequate for predicting the dynamic response of complex pathways. Hierarchical computational models that allow exploration of semi-quantitative dynamics are useful systems biology tools for theoreticians, experimentalists and clinicians and may provide a means for cross-communication. A novel approach for biological pathway modeling based on hybrid intelligent systems or soft computing technologies is presented here. Intelligent hybrid systems, which refers to several related computing methods such as fuzzy logic, neural nets, genetic algorithms, and statistical analysis, has become ubiquitous in engineering applications for complex control system modeling and design. Biological pathways may be considered to be complex control systems, which medicine tries to manipulate to achieve desired results. Thus, hybrid intelligent systems may provide a useful tool for modeling biological system dynamics and computational exploration of new drug targets. A new modeling approach based on these methods is presented in the context of hedgehog regulation of the cell cycle in granule cells. Code and input files can be found at the Bionet website: www.chip.ord/~wbosl/Software/Bionet. This paper presents the algorithmic methods needed for modeling complicated biochemical dynamics using rule-based models to represent expert knowledge in the context of cell cycle regulation and tumor growth. A notable feature of this modeling approach is that it allows biologists to build complex models from their knowledge base without the need to translate that knowledge into mathematical form. Dynamics on several levels, from molecular pathways to tissue growth, are seamlessly integrated. A number of common network motifs are examined and used to build a model of hedgehog regulation of the cell cycle in cerebellar neurons, which is believed to play a key role in the etiology of medulloblastoma, a devastating childhood brain cancer.

  9. Pathway collages: personalized multi-pathway diagrams.

    PubMed

    Paley, Suzanne; O'Maille, Paul E; Weaver, Daniel; Karp, Peter D

    2016-12-13

    Metabolic pathway diagrams are a classical way of visualizing a linked cascade of biochemical reactions. However, to understand some biochemical situations, viewing a single pathway is insufficient, whereas viewing the entire metabolic network results in information overload. How do we enable scientists to rapidly construct personalized multi-pathway diagrams that depict a desired collection of interacting pathways that emphasize particular pathway interactions? We define software for constructing personalized multi-pathway diagrams called pathway-collages using a combination of manual and automatic layouts. The user specifies a set of pathways of interest for the collage from a Pathway/Genome Database. Layouts for the individual pathways are generated by the Pathway Tools software, and are sent to a Javascript Pathway Collage application implemented using Cytoscape.js. That application allows the user to re-position pathways; define connections between pathways; change visual style parameters; and paint metabolomics, gene expression, and reaction flux data onto the collage to obtain a desired multi-pathway diagram. We demonstrate the use of pathway collages in two application areas: a metabolomics study of pathogen drug response, and an Escherichia coli metabolic model. Pathway collages enable facile construction of personalized multi-pathway diagrams.

  10. PathFinder: reconstruction and dynamic visualization of metabolic pathways.

    PubMed

    Goesmann, Alexander; Haubrock, Martin; Meyer, Folker; Kalinowski, Jörn; Giegerich, Robert

    2002-01-01

    Beyond methods for a gene-wise annotation and analysis of sequenced genomes new automated methods for functional analysis on a higher level are needed. The identification of realized metabolic pathways provides valuable information on gene expression and regulation. Detection of incomplete pathways helps to improve a constantly evolving genome annotation or discover alternative biochemical pathways. To utilize automated genome analysis on the level of metabolic pathways new methods for the dynamic representation and visualization of pathways are needed. PathFinder is a tool for the dynamic visualization of metabolic pathways based on annotation data. Pathways are represented as directed acyclic graphs, graph layout algorithms accomplish the dynamic drawing and visualization of the metabolic maps. A more detailed analysis of the input data on the level of biochemical pathways helps to identify genes and detect improper parts of annotations. As an Relational Database Management System (RDBMS) based internet application PathFinder reads a list of EC-numbers or a given annotation in EMBL- or Genbank-format and dynamically generates pathway graphs.

  11. The emerging complexity of ubiquitin architecture.

    PubMed

    Ohtake, Fumiaki; Tsuchiya, Hikaru

    2017-02-01

    Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  12. MEGADOCK-Web: an integrated database of high-throughput structure-based protein-protein interaction predictions.

    PubMed

    Hayashi, Takanori; Matsuzaki, Yuri; Yanagisawa, Keisuke; Ohue, Masahito; Akiyama, Yutaka

    2018-05-08

    Protein-protein interactions (PPIs) play several roles in living cells, and computational PPI prediction is a major focus of many researchers. The three-dimensional (3D) structure and binding surface are important for the design of PPI inhibitors. Therefore, rigid body protein-protein docking calculations for two protein structures are expected to allow elucidation of PPIs different from known complexes in terms of 3D structures because known PPI information is not explicitly required. We have developed rapid PPI prediction software based on protein-protein docking, called MEGADOCK. In order to fully utilize the benefits of computational PPI predictions, it is necessary to construct a comprehensive database to gather prediction results and their predicted 3D complex structures and to make them easily accessible. Although several databases exist that provide predicted PPIs, the previous databases do not contain a sufficient number of entries for the purpose of discovering novel PPIs. In this study, we constructed an integrated database of MEGADOCK PPI predictions, named MEGADOCK-Web. MEGADOCK-Web provides more than 10 times the number of PPI predictions than previous databases and enables users to conduct PPI predictions that cannot be found in conventional PPI prediction databases. In MEGADOCK-Web, there are 7528 protein chains and 28,331,628 predicted PPIs from all possible combinations of those proteins. Each protein structure is annotated with PDB ID, chain ID, UniProt AC, related KEGG pathway IDs, and known PPI pairs. Additionally, MEGADOCK-Web provides four powerful functions: 1) searching precalculated PPI predictions, 2) providing annotations for each predicted protein pair with an experimentally known PPI, 3) visualizing candidates that may interact with the query protein on biochemical pathways, and 4) visualizing predicted complex structures through a 3D molecular viewer. MEGADOCK-Web provides a huge amount of comprehensive PPI predictions based on docking calculations with biochemical pathways and enables users to easily and quickly assess PPI feasibilities by archiving PPI predictions. MEGADOCK-Web also promotes the discovery of new PPIs and protein functions and is freely available for use at http://www.bi.cs.titech.ac.jp/megadock-web/ .

  13. Competition for vitamin B1 (thiamin) structures numerous ecological interactions.

    PubMed

    Kraft, Clifford E; Angert, Esther R

    2017-06-01

    Thiamin (vitamin B1) is a cofactor required for essential biochemical reactions in all living organisms, yet free thiamin is scarce in the environment. The diversity of biochemical pathways involved in the acquisition, degradation, and synthesis of thiamin indicates that organisms have evolved numerous ecological strategies for meeting this nutritional requirement. In this review we synthesize information from multiple disciplines to show how the complex biochemistry of thiamin influences ecological outcomes of interactions between organisms in environments ranging from the open ocean and the Australian outback to the gastrointestinal tract of animals. We highlight population and ecosystem responses to the availability or absence of thiamin. These include widespread mortality of fishes, birds, and mammals, as well as the thiamin-dependent regulation of ocean productivity. Overall, we portray thiamin biochemistry as the foundation for molecularly mediated ecological interactions that influence survival and abundance of a vast array of organisms.

  14. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  15. Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

    PubMed

    Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

    2014-07-01

    Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

  16. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

    PubMed

    Bowman, John L; Kohchi, Takayuki; Yamato, Katsuyuki T; Jenkins, Jerry; Shu, Shengqiang; Ishizaki, Kimitsune; Yamaoka, Shohei; Nishihama, Ryuichi; Nakamura, Yasukazu; Berger, Frédéric; Adam, Catherine; Aki, Shiori Sugamata; Althoff, Felix; Araki, Takashi; Arteaga-Vazquez, Mario A; Balasubrmanian, Sureshkumar; Barry, Kerrie; Bauer, Diane; Boehm, Christian R; Briginshaw, Liam; Caballero-Perez, Juan; Catarino, Bruno; Chen, Feng; Chiyoda, Shota; Chovatia, Mansi; Davies, Kevin M; Delmans, Mihails; Demura, Taku; Dierschke, Tom; Dolan, Liam; Dorantes-Acosta, Ana E; Eklund, D Magnus; Florent, Stevie N; Flores-Sandoval, Eduardo; Fujiyama, Asao; Fukuzawa, Hideya; Galik, Bence; Grimanelli, Daniel; Grimwood, Jane; Grossniklaus, Ueli; Hamada, Takahiro; Haseloff, Jim; Hetherington, Alexander J; Higo, Asuka; Hirakawa, Yuki; Hundley, Hope N; Ikeda, Yoko; Inoue, Keisuke; Inoue, Shin-Ichiro; Ishida, Sakiko; Jia, Qidong; Kakita, Mitsuru; Kanazawa, Takehiko; Kawai, Yosuke; Kawashima, Tomokazu; Kennedy, Megan; Kinose, Keita; Kinoshita, Toshinori; Kohara, Yuji; Koide, Eri; Komatsu, Kenji; Kopischke, Sarah; Kubo, Minoru; Kyozuka, Junko; Lagercrantz, Ulf; Lin, Shih-Shun; Lindquist, Erika; Lipzen, Anna M; Lu, Chia-Wei; De Luna, Efraín; Martienssen, Robert A; Minamino, Naoki; Mizutani, Masaharu; Mizutani, Miya; Mochizuki, Nobuyoshi; Monte, Isabel; Mosher, Rebecca; Nagasaki, Hideki; Nakagami, Hirofumi; Naramoto, Satoshi; Nishitani, Kazuhiko; Ohtani, Misato; Okamoto, Takashi; Okumura, Masaki; Phillips, Jeremy; Pollak, Bernardo; Reinders, Anke; Rövekamp, Moritz; Sano, Ryosuke; Sawa, Shinichiro; Schmid, Marc W; Shirakawa, Makoto; Solano, Roberto; Spunde, Alexander; Suetsugu, Noriyuki; Sugano, Sumio; Sugiyama, Akifumi; Sun, Rui; Suzuki, Yutaka; Takenaka, Mizuki; Takezawa, Daisuke; Tomogane, Hirokazu; Tsuzuki, Masayuki; Ueda, Takashi; Umeda, Masaaki; Ward, John M; Watanabe, Yuichiro; Yazaki, Kazufumi; Yokoyama, Ryusuke; Yoshitake, Yoshihiro; Yotsui, Izumi; Zachgo, Sabine; Schmutz, Jeremy

    2017-10-05

    The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Walking the C4 pathway: past, present, and future.

    PubMed

    Furbank, Robert T

    2017-01-01

    The year 2016 marks 50 years since the publication of the seminal paper by Hatch and Slack describing the biochemical pathway we now know as C 4 photosynthesis. This review provides insight into the initial discovery of this pathway, the clues which led Hatch and Slack and others to these definitive experiments, some of the intrigue which surrounds the international activities which led up to the discovery, and personal insights into the future of this research field. While the biochemical understanding of the basic pathways came quickly, the role of the bundle sheath intermediate CO 2 pool was not understood for a number of years, and the nature of C 4 as a biochemical CO 2 pump then linked the unique Kranz anatomy of C 4 plants to their biochemical specialization. Decades of "grind and find biochemistry" and leaf physiology fleshed out the regulation of the pathway and the differences in physiological response to the environment between C 3 and C 4 plants. The more recent advent of plant transformation then high-throughput RNA and DNA sequencing and synthetic biology has allowed us both to carry out biochemical experiments and test hypotheses in planta and to better understand the evolution-driven molecular and genetic changes which occurred in the genomes of plants in the transition from C 3 to C 4 Now we are using this knowledge in attempts to engineer C 4 rice and improve the C 4 engine itself for enhanced food security and to provide novel biofuel feedstocks. The next 50 years of photosynthesis will no doubt be challenging, stimulating, and a drawcard for the best young minds in plant biology. © 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.

  18. Walking the C4 pathway: past, present, and future.

    PubMed

    Furbank, Robert T

    2016-07-01

    The year 2016 marks 50 years since the publication of the seminal paper by Hatch and Slack describing the biochemical pathway we now know as C4 photosynthesis. This review provides insight into the initial discovery of this pathway, the clues which led Hatch and Slack and others to these definitive experiments, some of the intrigue which surrounds the international activities which led up to the discovery, and personal insights into the future of this research field. While the biochemical understanding of the basic pathways came quickly, the role of the bundle sheath intermediate CO2 pool was not understood for a number of years, and the nature of C4 as a biochemical CO2 pump then linked the unique Kranz anatomy of C4 plants to their biochemical specialization. Decades of "grind and find biochemistry" and leaf physiology fleshed out the regulation of the pathway and the differences in physiological response to the environment between C3 and C4 plants. The more recent advent of plant transformation then high-throughput RNA and DNA sequencing and synthetic biology has allowed us both to carry out biochemical experiments and test hypotheses in planta and to better understand the evolution-driven molecular and genetic changes which occurred in the genomes of plants in the transition from C3 to C4 Now we are using this knowledge in attempts to engineer C4 rice and improve the C4 engine itself for enhanced food security and to provide novel biofuel feedstocks. The next 50 years of photosynthesis will no doubt be challenging, stimulating, and a drawcard for the best young minds in plant biology. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. Seasonal induction of alternative principal pathway for rose flower scent

    PubMed Central

    Hirata, Hiroshi; Ohnishi, Toshiyuki; Tomida, Kensuke; Ishida, Haruka; Kanda, Momoyo; Sakai, Miwa; Yoshimura, Jin; Suzuki, Hideyuki; Ishikawa, Takamasa; Dohra, Hideo; Watanabe, Naoharu

    2016-01-01

    Ecological adaptations to seasonal changes are often observed in the phenotypic traits of plants and animals, and these adaptations are usually expressed through the production of different biochemical end products. In this study, ecological adaptations are observed in a biochemical pathway without alteration of the end products. We present an alternative principal pathway to the characteristic floral scent compound 2-phenylethanol (2PE) in roses. The new pathway is seasonally induced in summer as a heat adaptation that uses rose phenylpyruvate decarboxylase (RyPPDC) as a novel enzyme. RyPPDC transcript levels and the resulting production of 2PE are increased time-dependently under high temperatures. The novel summer pathway produces levels of 2PE that are several orders of magnitude higher than those produced by the previously known pathway. Our results indicate that the alternative principal pathway identified here is a seasonal adaptation for managing the weakened volatility of summer roses. PMID:26831950

  20. Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.

    PubMed

    Cox, Nathan J; Unlu, Gokhan; Bisnett, Brittany J; Meister, Thomas R; Condon, Brett M; Luo, Peter M; Smith, Timothy J; Hanna, Michael; Chhetri, Abhishek; Soderblom, Erik J; Audhya, Anjon; Knapik, Ela W; Boyce, Michael

    2018-01-09

    The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

  1. Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer Allochromatium vinosum.

    PubMed

    Ghosh, Semanti; Bagchi, Angshuman

    2018-04-26

    Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.

  2. Glyphosate Resistance of C3 and C4 Weeds under Rising Atmospheric CO2

    PubMed Central

    Fernando, Nimesha; Manalil, Sudheesh; Florentine, Singarayer K.; Chauhan, Bhagirath S.; Seneweera, Saman

    2016-01-01

    The present paper reviews current knowledge on how changes of plant metabolism under elevated CO2 concentrations (e[CO2]) can affect the development of the glyphosate resistance of C3 and C4 weeds. Among the chemical herbicides, glyphosate, which is a non-selective and post-emergence herbicide, is currently the most widely used herbicide in global agriculture. As a consequence, glyphosate resistant weeds, particularly in major field crops, are a widespread problem and are becoming a significant challenge to future global food production. Of particular interest here it is known that the biochemical processes involved in photosynthetic pathways of C3 and C4 plants are different, which may have relevance to their competitive development under changing environmental conditions. It has already been shown that plant anatomical, morphological, and physiological changes under e[CO2] can be different, based on (i) the plant’s functional group, (ii) the available soil nutrients, and (iii) the governing water status. In this respect, C3 species are likely to have a major developmental advantage under a CO2 rich atmosphere, by being able to capitalize on the overall stimulatory effect of e[CO2]. For example, many tropical weed grass species fix CO2 from the atmosphere via the C4 photosynthetic pathway, which is a complex anatomical and biochemical variant of the C3 pathway. Thus, based on our current knowledge of CO2 fixing, it would appear obvious that the development of a glyphosate-resistant mechanism would be easier under an e[CO2] in C3 weeds which have a simpler photosynthetic pathway, than for C4 weeds. However, notwithstanding this logical argument, a better understanding of the biochemical, genetic, and molecular measures by which plants develop glyphosate resistance and how e[CO2] affects these measures will be important before attempting to innovate sustainable technology to manage the glyphosate-resistant evolution of weeds under e[CO2]. Such information will be of essential in managing weed control by herbicide use, and to thus ensure an increase in global food production in the event of increased atmospheric [CO2] levels. PMID:27446140

  3. Glyphosate Resistance of C3 and C4 Weeds under Rising Atmospheric CO2.

    PubMed

    Fernando, Nimesha; Manalil, Sudheesh; Florentine, Singarayer K; Chauhan, Bhagirath S; Seneweera, Saman

    2016-01-01

    The present paper reviews current knowledge on how changes of plant metabolism under elevated CO2 concentrations (e[CO2]) can affect the development of the glyphosate resistance of C3 and C4 weeds. Among the chemical herbicides, glyphosate, which is a non-selective and post-emergence herbicide, is currently the most widely used herbicide in global agriculture. As a consequence, glyphosate resistant weeds, particularly in major field crops, are a widespread problem and are becoming a significant challenge to future global food production. Of particular interest here it is known that the biochemical processes involved in photosynthetic pathways of C3 and C4 plants are different, which may have relevance to their competitive development under changing environmental conditions. It has already been shown that plant anatomical, morphological, and physiological changes under e[CO2] can be different, based on (i) the plant's functional group, (ii) the available soil nutrients, and (iii) the governing water status. In this respect, C3 species are likely to have a major developmental advantage under a CO2 rich atmosphere, by being able to capitalize on the overall stimulatory effect of e[CO2]. For example, many tropical weed grass species fix CO2 from the atmosphere via the C4 photosynthetic pathway, which is a complex anatomical and biochemical variant of the C3 pathway. Thus, based on our current knowledge of CO2 fixing, it would appear obvious that the development of a glyphosate-resistant mechanism would be easier under an e[CO2] in C3 weeds which have a simpler photosynthetic pathway, than for C4 weeds. However, notwithstanding this logical argument, a better understanding of the biochemical, genetic, and molecular measures by which plants develop glyphosate resistance and how e[CO2] affects these measures will be important before attempting to innovate sustainable technology to manage the glyphosate-resistant evolution of weeds under e[CO2]. Such information will be of essential in managing weed control by herbicide use, and to thus ensure an increase in global food production in the event of increased atmospheric [CO2] levels.

  4. Exploitation of Nontraditional Corp, Yacon, in Breast Cancer Prevention Using Preclinical Rat Model

    DTIC Science & Technology

    2011-07-01

    liver glucose disposal evident along sorbitol, PPP, and hexosamine pathways. • Gut microbiome : A significant impact of diet on levels of...biochemicals reflecting metabolism of the gut microbiome was evident in plasma and liver and observed for several classes of metabolites. Biochemicals...acid metabolites reflecting activity of the gut microbiome contribute to host metabolic pathways and/or must be metabolized further by the liver

  5. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

    PubMed Central

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-01-01

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. PMID:22508728

  6. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

    PubMed

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-04-15

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

  7. A directed-overflow and damage-control N -glycosidase in riboflavin biosynthesis

    DOE PAGES

    Frelin, Océane; Huang, Lili; Hasnain, Ghulam; ...

    2015-02-15

    Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multistep pathway. The early intermediates of this pathway are notoriously reactive, and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. Here we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA, respectively). We present cheminformatic, biochemical, genetic, and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis,more » yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multienzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. We find COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites.« less

  8. Modularization of biochemical networks based on classification of Petri net t-invariants.

    PubMed

    Grafahrend-Belau, Eva; Schreiber, Falk; Heiner, Monika; Sackmann, Andrea; Junker, Björn H; Grunwald, Stefanie; Speer, Astrid; Winder, Katja; Koch, Ina

    2008-02-08

    Structural analysis of biochemical networks is a growing field in bioinformatics and systems biology. The availability of an increasing amount of biological data from molecular biological networks promises a deeper understanding but confronts researchers with the problem of combinatorial explosion. The amount of qualitative network data is growing much faster than the amount of quantitative data, such as enzyme kinetics. In many cases it is even impossible to measure quantitative data because of limitations of experimental methods, or for ethical reasons. Thus, a huge amount of qualitative data, such as interaction data, is available, but it was not sufficiently used for modeling purposes, until now. New approaches have been developed, but the complexity of data often limits the application of many of the methods. Biochemical Petri nets make it possible to explore static and dynamic qualitative system properties. One Petri net approach is model validation based on the computation of the system's invariant properties, focusing on t-invariants. T-invariants correspond to subnetworks, which describe the basic system behavior.With increasing system complexity, the basic behavior can only be expressed by a huge number of t-invariants. According to our validation criteria for biochemical Petri nets, the necessary verification of the biological meaning, by interpreting each subnetwork (t-invariant) manually, is not possible anymore. Thus, an automated, biologically meaningful classification would be helpful in analyzing t-invariants, and supporting the understanding of the basic behavior of the considered biological system. Here, we introduce a new approach to automatically classify t-invariants to cope with network complexity. We apply clustering techniques such as UPGMA, Complete Linkage, Single Linkage, and Neighbor Joining in combination with different distance measures to get biologically meaningful clusters (t-clusters), which can be interpreted as modules. To find the optimal number of t-clusters to consider for interpretation, the cluster validity measure, Silhouette Width, is applied. We considered two different case studies as examples: a small signal transduction pathway (pheromone response pathway in Saccharomyces cerevisiae) and a medium-sized gene regulatory network (gene regulation of Duchenne muscular dystrophy). We automatically classified the t-invariants into functionally distinct t-clusters, which could be interpreted biologically as functional modules in the network. We found differences in the suitability of the various distance measures as well as the clustering methods. In terms of a biologically meaningful classification of t-invariants, the best results are obtained using the Tanimoto distance measure. Considering clustering methods, the obtained results suggest that UPGMA and Complete Linkage are suitable for clustering t-invariants with respect to the biological interpretability. We propose a new approach for the biological classification of Petri net t-invariants based on cluster analysis. Due to the biologically meaningful data reduction and structuring of network processes, large sets of t-invariants can be evaluated, allowing for model validation of qualitative biochemical Petri nets. This approach can also be applied to elementary mode analysis.

  9. Modularization of biochemical networks based on classification of Petri net t-invariants

    PubMed Central

    Grafahrend-Belau, Eva; Schreiber, Falk; Heiner, Monika; Sackmann, Andrea; Junker, Björn H; Grunwald, Stefanie; Speer, Astrid; Winder, Katja; Koch, Ina

    2008-01-01

    Background Structural analysis of biochemical networks is a growing field in bioinformatics and systems biology. The availability of an increasing amount of biological data from molecular biological networks promises a deeper understanding but confronts researchers with the problem of combinatorial explosion. The amount of qualitative network data is growing much faster than the amount of quantitative data, such as enzyme kinetics. In many cases it is even impossible to measure quantitative data because of limitations of experimental methods, or for ethical reasons. Thus, a huge amount of qualitative data, such as interaction data, is available, but it was not sufficiently used for modeling purposes, until now. New approaches have been developed, but the complexity of data often limits the application of many of the methods. Biochemical Petri nets make it possible to explore static and dynamic qualitative system properties. One Petri net approach is model validation based on the computation of the system's invariant properties, focusing on t-invariants. T-invariants correspond to subnetworks, which describe the basic system behavior. With increasing system complexity, the basic behavior can only be expressed by a huge number of t-invariants. According to our validation criteria for biochemical Petri nets, the necessary verification of the biological meaning, by interpreting each subnetwork (t-invariant) manually, is not possible anymore. Thus, an automated, biologically meaningful classification would be helpful in analyzing t-invariants, and supporting the understanding of the basic behavior of the considered biological system. Methods Here, we introduce a new approach to automatically classify t-invariants to cope with network complexity. We apply clustering techniques such as UPGMA, Complete Linkage, Single Linkage, and Neighbor Joining in combination with different distance measures to get biologically meaningful clusters (t-clusters), which can be interpreted as modules. To find the optimal number of t-clusters to consider for interpretation, the cluster validity measure, Silhouette Width, is applied. Results We considered two different case studies as examples: a small signal transduction pathway (pheromone response pathway in Saccharomyces cerevisiae) and a medium-sized gene regulatory network (gene regulation of Duchenne muscular dystrophy). We automatically classified the t-invariants into functionally distinct t-clusters, which could be interpreted biologically as functional modules in the network. We found differences in the suitability of the various distance measures as well as the clustering methods. In terms of a biologically meaningful classification of t-invariants, the best results are obtained using the Tanimoto distance measure. Considering clustering methods, the obtained results suggest that UPGMA and Complete Linkage are suitable for clustering t-invariants with respect to the biological interpretability. Conclusion We propose a new approach for the biological classification of Petri net t-invariants based on cluster analysis. Due to the biologically meaningful data reduction and structuring of network processes, large sets of t-invariants can be evaluated, allowing for model validation of qualitative biochemical Petri nets. This approach can also be applied to elementary mode analysis. PMID:18257938

  10. SAR202 Genomes from the Dark Ocean Predict Pathways for the Oxidation of Recalcitrant Dissolved Organic Matter.

    PubMed

    Landry, Zachary; Swan, Brandon K; Herndl, Gerhard J; Stepanauskas, Ramunas; Giovannoni, Stephen J

    2017-04-18

    Deep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist. IMPORTANCE Carbon in the ocean is massively sequestered in a complex mixture of biologically refractory molecules that accumulate as the chemical end member of biological oxidation and diagenetic change. However, few details are known about the biochemical machinery of carbon sequestration in the deep ocean. Reconstruction of the metabolism of a deep-ocean microbial clade, SAR202, led to postulation of new biochemical pathways that may be the penultimate stages of DOM oxidation to refractory forms that persist. These pathways are tied to a proliferation of oxidative enzymes. This research illuminates dark-ocean biochemistry that is broadly consequential for reconstructing the global carbon cycle. Copyright © 2017 Landry et al.

  11. Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

    PubMed

    Schiffmann, D A; White, J H; Cooper, A; Nutley, M A; Harding, S E; Jumel, K; Solari, R; Ray, K P; Gay, N J

    1999-09-07

    In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

  12. Evolutionary innovation and diversification of carotenoid-based pigmentation in finches.

    PubMed

    Ligon, Russell A; Simpson, Richard K; Mason, Nicholas A; Hill, Geoffrey E; McGraw, Kevin J

    2016-12-01

    The ornaments used by animals to mediate social interactions are diverse, and by reconstructing their evolutionary pathways we can gain new insights into the mechanisms underlying ornamental innovation and variability. Here, we examine variation in plumage carotenoids among the true finches (Aves: Fringillidae) using biochemical and comparative phylogenetic analyses to reconstruct the evolutionary history of carotenoid states and evaluate competing models of carotenoid evolution. Our comparative analyses reveal that the most likely ancestor of finches used dietary carotenoids as yellow plumage colorants, and that the ability to metabolically modify dietary carotenoids into more complex pigments arose secondarily once finches began to use modified carotenoids to create red plumage. Following the evolutionary "innovation" that enabled modified red carotenoid pigments to be deposited as plumage colorants, many finch species subsequently modified carotenoid biochemical pathways to create yellow plumage. However, no reversions to dietary carotenoids were observed. The finding that ornaments and their underlying mechanisms may be operating under different selection regimes-where ornamental trait colors undergo frequent reversions (e.g., between red and yellow plumage) while carotenoid metabolization mechanisms are more conserved-supports a growing empirical framework suggesting different evolutionary patterns for ornaments and the mechanistic innovations that facilitate their diversification. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

  13. Temporal and fluoride control of secondary metabolism regulates cellular organofluorine biosynthesis

    PubMed Central

    Walker, Mark C.; Wen, Miao; Weeks, Amy M.; Chang, Michelle C. Y.

    2018-01-01

    Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into probe or therapeutic molecules. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya’s resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host’s metabolic machinery to discriminate between fluorinated and non–fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl–CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products. PMID:22769062

  14. Modeling the optimal central carbon metabolic pathways under feedback inhibition using flux balance analysis.

    PubMed

    De, Rajat K; Tomar, Namrata

    2012-12-01

    Metabolism is a complex process for energy production for cellular activity. It consists of a cascade of reactions that form a highly branched network in which the product of one reaction is the reactant of the next reaction. Metabolic pathways efficiently produce maximal amount of biomass while maintaining a steady-state behavior. The steady-state activity of such biochemical pathways necessarily incorporates feedback inhibition of the enzymes. This observation motivates us to incorporate feedback inhibition for modeling the optimal activity of metabolic pathways using flux balance analysis (FBA). We demonstrate the effectiveness of the methodology on a synthetic pathway with and without feedback inhibition. Similarly, for the first time, the Central Carbon Metabolic (CCM) pathways of Saccharomyces cerevisiae and Homo sapiens have been modeled and compared based on the above understanding. The optimal pathway, which maximizes the amount of the target product(s), is selected from all those obtained by the proposed method. For this, we have observed the concentration of the product inhibited enzymes of CCM pathway and its influence on its corresponding metabolite/substrate. We have also studied the concentration of the enzymes which are responsible for the synthesis of target products. We further hypothesize that an optimal pathway would opt for higher flux rate reactions. In light of these observations, we can say that an optimal pathway should have lower enzyme concentration and higher flux rates. Finally, we demonstrate the superiority of the proposed method by comparing it with the extreme pathway analysis.

  15. Biomass recalcitrance: a multi-scale, multi-factor, and conversion-specific property.

    PubMed

    McCann, Maureen C; Carpita, Nicholas C

    2015-07-01

    Recalcitrance of plant biomass to enzymatic hydrolysis for biofuel production is thought to be a property conferred by lignin or lignin-carbohydrate complexes. However, chemical catalytic and thermochemical conversion pathways, either alone or in combination with biochemical and fermentative pathways, now provide avenues to utilize lignin and to expand the product range beyond ethanol or butanol. To capture all of the carbon in renewable biomass, both lignin-derived aromatics and polysaccharide-derived sugars need to be transformed by catalysts to liquid hydrocarbons and high-value co-products. We offer a new definition of recalcitrance as those features of biomass which disproportionately increase energy requirements in conversion processes, increase the cost and complexity of operations in the biorefinery, and/or reduce the recovery of biomass carbon into desired products. The application of novel processing technologies applied to biomass reveal new determinants of recalcitrance that comprise a broad range of molecular, nanoscale, and macroscale factors. Sampling natural genetic diversity within a species, transgenic approaches, and synthetic biology approaches are all strategies that can be used to select biomass for reduced recalcitrance in various pretreatments and conversion pathways. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Progress in understanding the neuronal SNARE function and its regulation.

    PubMed

    Yoon, T-Y; Shin, Y-K

    2009-02-01

    Vesicle budding and fusion underlies many essential biochemical deliveries in eukaryotic cells, and its core fusion machinery is thought to be built on one protein family named soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Recent technical advances based on site-directed fluorescence labelling and nano-scale detection down to the single-molecule level rapidly unveiled the protein and the lipid intermediates along the fusion pathway as well as the molecular actions of fusion effectors. Here we summarize these new exciting findings in context with a new mechanistic model that reconciles two existing fusion models: the proteinaceous pore model and the hemifusion model. Further, we attempt to locate the points of action for the fusion effectors along the fusion pathway and to delineate the energetic interplay between the SNARE complexes and the fusion effectors.

  17. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

    PubMed Central

    Osinski, Adam; Tomchick, Diana R; Brautigam, Chad A

    2017-01-01

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex. PMID:29063833

  18. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

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

    Bae, Sung Jun; Ni, Lisheng; Osinski, Adam

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1more » directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.« less

  19. Systems Biology Graphical Notation: Entity Relationship language Level 1 Version 2.

    PubMed

    Sorokin, Anatoly; Le Novère, Nicolas; Luna, Augustin; Czauderna, Tobias; Demir, Emek; Haw, Robin; Mi, Huaiyu; Moodie, Stuart; Schreiber, Falk; Villéger, Alice

    2015-09-04

    The Systems Biological Graphical Notation (SBGN) is an international community effort for standardized graphical representations of biological pathways and networks. The goal of SBGN is to provide unambiguous pathway and network maps for readers with different scientific backgrounds as well as to support efficient and accurate exchange of biological knowledge between different research communities, industry, and other players in systems biology. Three SBGN languages, Process Description (PD), Entity Relationship (ER) and Activity Flow (AF), allow for the representation of different aspects of biological and biochemical systems at different levels of detail. The SBGN Entity Relationship language (ER) represents biological entities and their interactions and relationships within a network. SBGN ER focuses on all potential relationships between entities without considering temporal aspects. The nodes (elements) describe biological entities, such as proteins and complexes. The edges (connections) provide descriptions of interactions and relationships (or influences), e.g., complex formation, stimulation and inhibition. Among all three languages of SBGN, ER is the closest to protein interaction networks in biological literature and textbooks, but its well-defined semantics offer a superior precision in expressing biological knowledge.

  20. Pharmacophore based design of some multi-targeted compounds targeted against pathways of diabetic complications.

    PubMed

    Chadha, Navriti; Silakari, Om

    2017-09-01

    Diabetic complications is a complex metabolic disorder developed primarily due to prolonged hyperglycemia in the body. The complexity of the disease state as well as the unifying pathophysiology discussed in the literature reports exhibited that the use of multi-targeted agents with multiple complementary biological activities may offer promising therapy for the intervention of the disease over the single-target drugs. In the present study, novel thiazolidine-2,4-dione analogues were designed as multi-targeted agents implicated against the molecular pathways involved in diabetic complications using knowledge based as well as in-silico approaches such as pharmacophore mapping, molecular docking etc. The hit molecules were duly synthesized and biochemical estimation of these molecules against aldose reductase (ALR2), protein kinase Cβ (PKCβ) and poly (ADP-ribose) polymerase 1 (PARP-1) led to identification of compound 2 that showed good potency against PARP-1 and ALR2 enzymes. These positive results support the progress of a low cost multi-targeted agent with putative roles in diabetic complications. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Ras plasma membrane signalling platforms

    PubMed Central

    2005-01-01

    The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction. PMID:15954863

  2. The microbial nitrogen-cycling network.

    PubMed

    Kuypers, Marcel M M; Marchant, Hannah K; Kartal, Boran

    2018-05-01

    Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet. By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth. The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms. In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications.

  3. 13C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production

    PubMed Central

    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang

    2015-01-01

    Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, 13C Metabolic Flux Analysis (13C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many 13C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of 13C-MFA and illustrate how 13C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms PMID:28952565

  4. Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER).

    PubMed

    Wolz, Olaf-Oliver; Koegl, Manfred; Weber, Alexander N R

    2018-01-01

    Recent structural, biochemical, and functional studies have led to the notion that many of the post-receptor signaling complexes in innate immunity have a multimeric, multi-protein architecture whose hierarchical assembly is vital for function. The Myddosome is a post-receptor complex in the cytoplasmic signaling of Toll-like receptors (TLR) and the Interleukin-1 receptor (IL-1R), involving the proteins MyD88, IL-1R-associated kinase 4 (IRAK4), and IRAK2. Its importance is strikingly illustrated by the fact that rare germline mutations in MYD88 causing high susceptibility to infections are characterized by failure to assemble Myddosomes; conversely, gain-of-function MYD88 mutations leading to oncogenic hyperactivation of NF-κB show increased Myddosome formation. Reliable methods to probe Myddosome formation experimentally are therefore vital to further study the properties of this important post-receptor complex and its role in innate immunity, such as its regulation by posttranslational modification. Compared to structural and biochemical analyses, luminescence-based mammalian interactome mapping (LUMIER) is a straightforward, automatable, quantifiable, and versatile technique to study protein-protein interactions in a physiologically relevant context. We adapted LUMIER for Myddosome analysis and provide here a basic background of this technique, suitable experimental protocols, and its potential for medium-throughput screening. The principles presented herein can be adapted to other signaling pathways.

  5. Developing molecular tools for Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Noor-Mohammadi, Samaneh

    Microalgae have garnered increasing interest over the years for their ability to produce compounds ranging from biofuels to neutraceuticals. A main focus of researchers has been to use microalgae as a natural bioreactor for the production of valuable and complex compounds. Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. To take full advantage of these organisms' natural abilities, sophisticated molecular tools are needed to be able to introduce and functionally express multiple gene biosynthetic pathways in its genome. To achieve the above objective, we have sought to establish a method to construct, integrate and express multigene operons in the chloroplast and nuclear genome of the model microalgae Chlamydomonas reinhardtii. Here we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast, or by random integration in the nuclear genome of C. reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii and up to three reporter proteins (Ble, AphVIII, and GFP) in its nuclear genome. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated-region selection when constructing a target pathway. In addition, this work focuses on expressing the cofactor regeneration enzyme phosphite dehydrogenase (PTDH) in the chloroplast and nuclear genomes of C. reinhardtii. The PTDH enzyme converts phosphite into phosphate and NAD(P)+ into NAD(P)H. The reduced nicotinamide cofactor NAD(P)H plays a pivotal role in many biochemical oxidation and reduction reactions, thus this enzyme would allow regeneration of NAD(P)H in a microalgae strain over-expressing a NAD(P)H-dependent oxidoreductase. A phosphite dehydrogenase gene was introduced into the chloroplast genome (codon optimized) and nuclear genome of C. reinhardtii by biolistic transformation and electroporation in separate events, respectively. Successful expression of the heterologous protein was confirmed by transcript analysis and protein analysis. In conclusion, this new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast or nuclear genome of microalgae, and this should aid current efforts to engineer algae for recombinant protein expression, biofuels production and production of other desirable natural products.

  6. Plasma adiponectin complexes have distinct biochemical characteristics.

    PubMed

    Schraw, Todd; Wang, Zhao V; Halberg, Nils; Hawkins, Meredith; Scherer, Philipp E

    2008-05-01

    Adipocytes release the secretory protein adiponectin in a number of different higher-order complexes. Once synthesized and assembled in the secretory pathway of the adipocyte, these complexes circulate as biochemically distinct and stable entities with little evidence of interchange between the different forms that include a high-molecular-weight (HMW) species, a hexamer (low-molecular-weight form), and a trimeric form of the complexes. Here, we validate a high-resolution gel filtration method that reproducibly separates the three complexes in recombinant adiponectin and adiponectin from human and murine samples. We demonstrate that the HMW form is prominently reduced in male vs. female subjects and in obese, insulin-resistant vs. lean, insulin-sensitive individuals. A direct comparison of human and mouse adiponectin demonstrates that the trimer is generally more abundant in human serum. Furthermore, when the production of adiponectin is reduced, either by obesity or in mice carrying only a single functional allele of the adiponectin locus, then the amount of the HMW form is selectively reduced in circulation. The complex distribution of adiponectin can be regulated in several ways. Both mouse and human HMW adiponectin are very stable under basic conditions but are exquisitely labile under acidic conditions below pH 7. Murine and human adiponectin HMW forms also display differential susceptibility to the presence of calcium in the buffer. A mutant form of adiponectin unable to bind calcium is less susceptible to changes in calcium concentrations. However, the lack of calcium binding results in a destabilization of the structure. Disulfide bond formation (at position C39) is also important for complex formation. A mutant form of adiponectin lacking C39 prominently forms HMW and trimer but not the low-molecular-weight form. Injection of adiponectin with a fluorescent label reveals that over time, the various complexes do not interconvert in vivo. The stability of adiponectin complexes highlights that the production and secretion of these forms from fat cells has a major influence on the circulating levels of each complex.

  7. Activity-based protein profiling for biochemical pathway discovery in cancer

    PubMed Central

    Nomura, Daniel K.; Dix, Melissa M.; Cravatt, Benjamin F.

    2011-01-01

    Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment. PMID:20703252

  8. Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts1[C][W][OA

    PubMed Central

    Hennen-Bierwagen, Tracie A.; Lin, Qiaohui; Grimaud, Florent; Planchot, Véronique; Keeling, Peter L.; James, Martha G.; Myers, Alan M.

    2009-01-01

    Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds. PMID:19168640

  9. Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

    PubMed

    Scrima, Andrea; Fischer, Eric S; Lingaraju, Gondichatnahalli M; Böhm, Kerstin; Cavadini, Simone; Thomä, Nicolas H

    2011-09-16

    The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Cellular compartmentalization of secondary metabolism

    USDA-ARS?s Scientific Manuscript database

    Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors sh...

  11. Alterations in metabolic pathways and networks in Alzheimer's disease

    PubMed Central

    Kaddurah-Daouk, R; Zhu, H; Sharma, S; Bogdanov, M; Rozen, S G; Matson, W; Oki, N O; Motsinger-Reif, A A; Churchill, E; Lei, Z; Appleby, D; Kling, M A; Trojanowski, J Q; Doraiswamy, P M; Arnold, S E

    2013-01-01

    The pathogenic mechanisms of Alzheimer's disease (AD) remain largely unknown and clinical trials have not demonstrated significant benefit. Biochemical characterization of AD and its prodromal phase may provide new diagnostic and therapeutic insights. We used targeted metabolomics platform to profile cerebrospinal fluid (CSF) from AD (n=40), mild cognitive impairment (MCI, n=36) and control (n=38) subjects; univariate and multivariate analyses to define between-group differences; and partial least square-discriminant analysis models to classify diagnostic groups using CSF metabolomic profiles. A partial correlation network was built to link metabolic markers, protein markers and disease severity. AD subjects had elevated methionine (MET), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid, xanthosine and glutathione versus controls. MCI subjects had elevated 5-HIAA, MET, hypoxanthine and other metabolites versus controls. Metabolite ratios revealed changes within tryptophan, MET and purine pathways. Initial pathway analyses identified steps in several pathways that appear altered in AD and MCI. A partial correlation network showed total tau most directly related to norepinephrine and purine pathways; amyloid-β (Ab42) was related directly to an unidentified metabolite and indirectly to 5-HIAA and MET. These findings indicate that MCI and AD are associated with an overlapping pattern of perturbations in tryptophan, tyrosine, MET and purine pathways, and suggest that profound biochemical alterations are linked to abnormal Ab42 and tau metabolism. Metabolomics provides powerful tools to map interlinked biochemical pathway perturbations and study AD as a disease of network failure. PMID:23571809

  12. Alterations in metabolic pathways and networks in Alzheimer's disease.

    PubMed

    Kaddurah-Daouk, R; Zhu, H; Sharma, S; Bogdanov, M; Rozen, S G; Matson, W; Oki, N O; Motsinger-Reif, A A; Churchill, E; Lei, Z; Appleby, D; Kling, M A; Trojanowski, J Q; Doraiswamy, P M; Arnold, S E

    2013-04-09

    The pathogenic mechanisms of Alzheimer's disease (AD) remain largely unknown and clinical trials have not demonstrated significant benefit. Biochemical characterization of AD and its prodromal phase may provide new diagnostic and therapeutic insights. We used targeted metabolomics platform to profile cerebrospinal fluid (CSF) from AD (n=40), mild cognitive impairment (MCI, n=36) and control (n=38) subjects; univariate and multivariate analyses to define between-group differences; and partial least square-discriminant analysis models to classify diagnostic groups using CSF metabolomic profiles. A partial correlation network was built to link metabolic markers, protein markers and disease severity. AD subjects had elevated methionine (MET), 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid, xanthosine and glutathione versus controls. MCI subjects had elevated 5-HIAA, MET, hypoxanthine and other metabolites versus controls. Metabolite ratios revealed changes within tryptophan, MET and purine pathways. Initial pathway analyses identified steps in several pathways that appear altered in AD and MCI. A partial correlation network showed total tau most directly related to norepinephrine and purine pathways; amyloid-β (Ab42) was related directly to an unidentified metabolite and indirectly to 5-HIAA and MET. These findings indicate that MCI and AD are associated with an overlapping pattern of perturbations in tryptophan, tyrosine, MET and purine pathways, and suggest that profound biochemical alterations are linked to abnormal Ab42 and tau metabolism. Metabolomics provides powerful tools to map interlinked biochemical pathway perturbations and study AD as a disease of network failure.

  13. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    PubMed Central

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  14. Metabolomic strategies to map functions of metabolic pathways

    PubMed Central

    Mulvihill, Melinda M.

    2014-01-01

    Genome sequencing efforts have revealed a strikingly large number of unannotated and uncharacterized genes that fall into metabolic enzymes classes, likely indicating that our current knowledge of biochemical pathways in normal physiology, let alone in disease states, remains largely incomplete. This realization presents a daunting challenge for post-genomic-era scientists in deciphering the biochemical and (patho)physiological roles of these enzymes and their metabolites and metabolic networks. This is further complicated by many recent studies showing a rewiring of normal metabolic networks in disease states to give rise to unique pathophysiological functions of enzymes, metabolites, and metabolic pathways. This review focuses on recent discoveries made using metabolic mapping technologies to uncover novel pathways and metabolite-mediated posttranslational modifications and epigenetic alterations and their impact on physiology and disease. PMID:24918200

  15. Metatranscriptomic analysis of diverse microbial communities reveals core metabolic pathways and microbiome-specific functionality.

    PubMed

    Jiang, Yue; Xiong, Xuejian; Danska, Jayne; Parkinson, John

    2016-01-12

    Metatranscriptomics is emerging as a powerful technology for the functional characterization of complex microbial communities (microbiomes). Use of unbiased RNA-sequencing can reveal both the taxonomic composition and active biochemical functions of a complex microbial community. However, the lack of established reference genomes, computational tools and pipelines make analysis and interpretation of these datasets challenging. Systematic studies that compare data across microbiomes are needed to demonstrate the ability of such pipelines to deliver biologically meaningful insights on microbiome function. Here, we apply a standardized analytical pipeline to perform a comparative analysis of metatranscriptomic data from diverse microbial communities derived from mouse large intestine, cow rumen, kimchi culture, deep-sea thermal vent and permafrost. Sequence similarity searches allowed annotation of 19 to 76% of putative messenger RNA (mRNA) reads, with the highest frequency in the kimchi dataset due to its relatively low complexity and availability of closely related reference genomes. Metatranscriptomic datasets exhibited distinct taxonomic and functional signatures. From a metabolic perspective, we identified a common core of enzymes involved in amino acid, energy and nucleotide metabolism and also identified microbiome-specific pathways such as phosphonate metabolism (deep sea) and glycan degradation pathways (cow rumen). Integrating taxonomic and functional annotations within a novel visualization framework revealed the contribution of different taxa to metabolic pathways, allowing the identification of taxa that contribute unique functions. The application of a single, standard pipeline confirms that the rich taxonomic and functional diversity observed across microbiomes is not simply an artefact of different analysis pipelines but instead reflects distinct environmental influences. At the same time, our findings show how microbiome complexity and availability of reference genomes can impact comprehensive annotation of metatranscriptomes. Consequently, beyond the application of standardized pipelines, additional caution must be taken when interpreting their output and performing downstream, microbiome-specific, analyses. The pipeline used in these analyses along with a tutorial has been made freely available for download from our project website: http://www.compsysbio.org/microbiome .

  16. Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli.

    PubMed

    Song, Hun-Suk; Jeon, Jong-Min; Kim, Hyun-Joong; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Kim, Junyoung; Won Hong, Ju; Gi Hong, Yoon; Young Choi, Kwon; Kim, Yun-Gon; Kim, Wooseong; Yang, Yung-Hun

    2017-12-01

    To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD + , was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

    PubMed

    Hartuti, Endah Dwi; Inaoka, Daniel Ken; Komatsuya, Keisuke; Miyazaki, Yukiko; Miller, Russell J; Xinying, Wang; Sadikin, Mohamad; Prabandari, Erwahyuni Endang; Waluyo, Danang; Kuroda, Marie; Amalia, Eri; Matsuo, Yuichi; Nugroho, Nuki B; Saimoto, Hiroyuki; Pramisandi, Amila; Watanabe, Yoh-Ichi; Mori, Mihoko; Shiomi, Kazuro; Balogun, Emmanuel Oluwadare; Shiba, Tomoo; Harada, Shigeharu; Nozaki, Tomoyoshi; Kita, Kiyoshi

    2018-03-01

    Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc 1 complex inhibitor. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Effects of Acute Stress on Thrombosis.

    PubMed

    Bentur, Ohad S; Sarig, Galit; Brenner, Benjamin; Jacob, Giris

    2018-06-18

    Stress, the nonspecific response to any demand for change, is an adaptive response of the human body to various stimulants. As such, stress-induced hypercoagulation may represent an adaptive response to bleeding. Numerous epidemiological studies have revealed that a correlation exists between stress and thrombotic risk and biochemically, links of the relationship between psychological stress and coagulation pathways have been made. The stress reaction is coupled with neurohormonal changes mediated mainly by the sympathetic neural system and the hypothalamic-pituitary-adrenal axis. Singling out the specific pathways affecting coagulation in this complex response is hampered by many confounders. The mediators of the stress reaction (neurotransmitters and hormones) can directly affect platelets and the coagulation cascade and indirectly affect hemostasis via changes in hemodynamics. In this review, the authors will delineate the distinct neurobiological mechanisms that govern the effects of stress on coagulation, and report their recent findings. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  19. Cortical Actomyosin Breakage Triggers Shape Oscillations in Cells and Cell Fragments

    PubMed Central

    Paluch, Ewa; Piel, Matthieu; Prost, Jacques; Bornens, Michel; Sykes, Cécile

    2005-01-01

    Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization. PMID:15879479

  20. Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

    PubMed Central

    Coluccio, Alison; Bogengruber, Edith; Conrad, Michael N.; Dresser, Michael E.; Briza, Peter; Neiman, Aaron M.

    2004-01-01

    The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure. PMID:15590821

  1. Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during sevenless signaling.

    PubMed

    Herbst, R; Carroll, P M; Allard, J D; Schilling, J; Raabe, T; Simon, M A

    1996-06-14

    The SH2 domain-containing phosphotyrosine phosphatase Corkscrew (CSW) is an essential component of the signaling pathway initiated by the activation of the sevenless receptor tyrosine kinase (SEV) during Drosophila eye development. We have used genetic and biochemical approaches to identify a substrate for CSW. Expression of a catalytically inactive CSW was used to trap CSW in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations which enhance the phenotype caused by overexpression of inactive CSW during photoreceptor development. Analysis of dos mutations indicates that DOS is a positive component of the SEV signaling pathway and suggests that DOS dephosphorylation by CSW may be a key event during signaling by SEV.

  2. A biochemical approach to identifying microRNA targets

    PubMed Central

    Karginov, Fedor V.; Conaco, Cecilia; Xuan, Zhenyu; Schmidt, Bryan H.; Parker, Joel S.; Mandel, Gail; Hannon, Gregory J.

    2007-01-01

    Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway. PMID:18042700

  3. Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain.

    PubMed

    Francki, Michael G; Hayton, Sarah; Gummer, Joel P A; Rawlinson, Catherine; Trengove, Robert D

    2016-02-01

    Metabolomics is becoming an increasingly important tool in plant genomics to decipher the function of genes controlling biochemical pathways responsible for trait variation. Although theoretical models can integrate genes and metabolites for trait variation, biological networks require validation using appropriate experimental genetic systems. In this study, we applied an untargeted metabolite analysis to mature grain of wheat homoeologous group 3 ditelosomic lines, selected compounds that showed significant variation between wheat lines Chinese Spring and at least one ditelosomic line, tracked the genes encoding enzymes of their biochemical pathway using the wheat genome survey sequence and determined the genetic components underlying metabolite variation. A total of 412 analytes were resolved in the wheat grain metabolome, and principal component analysis indicated significant differences in metabolite profiles between Chinese Spring and each ditelosomic lines. The grain metabolome identified 55 compounds positively matched against a mass spectral library where the majority showed significant differences between Chinese Spring and at least one ditelosomic line. Trehalose and branched-chain amino acids were selected for detailed investigation, and it was expected that if genes encoding enzymes directly related to their biochemical pathways were located on homoeologous group 3 chromosomes, then corresponding ditelosomic lines would have a significant reduction in metabolites compared with Chinese Spring. Although a proportion showed a reduction, some lines showed significant increases in metabolites, indicating that genes directly and indirectly involved in biosynthetic pathways likely regulate the metabolome. Therefore, this study demonstrated that wheat aneuploid lines are suitable experimental genetic system to validate metabolomics-genomics networks. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Complexity and performance of on-chip biochemical assays

    NASA Astrophysics Data System (ADS)

    Kopf-Sill, Anne R.; Nikiforov, Theo; Bousse, Luc J.; Nagle, Rob; Parce, J. W.

    1997-03-01

    The use of microchips for performing biochemical processes has the potential to reduce reagent use and thus assay costs, increase throughput, and automate complex processes. We are building a multifunctional platform that provides sensing and actuation functions for a variety of microchip- based biochemical and analytical processes. Here we describe recent experiments that include on-chip dilution, reagent mixing, reaction, separation, and detection for important classes of biochemical assays. Issues in chip design and control are discussed.

  5. Recovering metabolic pathways via optimization.

    PubMed

    Beasley, John E; Planes, Francisco J

    2007-01-01

    A metabolic pathway is a coherent set of enzyme catalysed biochemical reactions by which a living organism transforms an initial (source) compound into a final (target) compound. Some of the different metabolic pathways adopted within organisms have been experimentally determined. In this paper, we show that a number of experimentally determined metabolic pathways can be recovered by a mathematical optimization model.

  6. Plant MetGenMAP: an integrative analysis system for plant systems biology

    USDA-ARS?s Scientific Manuscript database

    We have developed a web-based system, Plant MetGenMAP, which can identify significantly altered biochemical pathways and highly affected biological processes, predict functional roles of pathway genes, and potential pathway-related regulatory motifs from transcript and metabolite profile datasets. P...

  7. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    PubMed

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

  8. Urine metabolomics.

    PubMed

    Zhang, Aihua; Sun, Hui; Wu, Xiuhong; Wang, Xijun

    2012-12-24

    Metabolomics is a powerful technique for the discovery of novel biomarkers and elucidation of biochemical pathways to improve diagnosis, prognosis and therapy. An advantage of this approach is its ability to assess global metabolic profiles to enhance pathologic characterization. Urine is an ideal bio-medium for disease study because it is readily available, easily obtained and less complex than other body fluids. Ease of collection allows for serial sampling to monitor disease and therapeutic response. Because of this potential, this paper will review urine metabolomic analysis, discuss its significance in the post-genomic era and highlight the specific roles of endogenous small molecule metabolites in this emerging field. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Smell of Stress: Identification of Induced Biochemical Pathways Affecting the Volatile Composition and Flavor Quality of Crops.

    PubMed

    Wüst, Matthias

    2018-04-11

    Recent research has shown that the biosynthesis of several key odorants is controlled by genes whose expression is altered or even induced by biotic or abiotic stress. These new findings provide a roadmap for improvement of flavor quality by the application of moderate, well-controlled stress. This strategy aims at reducing the flavor deficiencies in modern commercial varieties as a "green" alternative to genetic engineering. The workflow for a successful implementation of this approach, from the identification of key odorants by molecular science techniques to the investigation of mechanisms controlling their biosynthesis, is complex and calls for interdisciplinary research.

  10. Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

    PubMed

    Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

    2006-01-01

    The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

  11. New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD(+) salvage from nicotinamide.

    PubMed

    Dong, Wei-Ren; Sun, Cen-Cen; Zhu, Guan; Hu, Shi-Hua; Xiang, Li-Xin; Shao, Jian-Zhong

    2014-02-08

    In an effort to reconstitute the NAD(+) synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD(+)de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD(+) de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD(+) metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD(+) biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD(+) salvage pathway from nicotinamide. Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD(+) salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD(+) salvage pathway might be significant in some bacteria lacking NAD(+) de novo and NAD(+) salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD(+). However, this speculation needs to be experimentally tested.

  12. Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

    PubMed Central

    Scullion, Paul; del Pino, Ricardo C.; Vincent, Isabel M.; Zhang, Yong-Kang; Alley, Michael R. K.; Jacobs, Robert T.; Read, Kevin D.

    2018-01-01

    Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds. PMID:29425238

  13. Metabolomic strategies to map functions of metabolic pathways.

    PubMed

    Mulvihill, Melinda M; Nomura, Daniel K

    2014-08-01

    Genome sequencing efforts have revealed a strikingly large number of unannotated and uncharacterized genes that fall into metabolic enzymes classes, likely indicating that our current knowledge of biochemical pathways in normal physiology, let alone in disease states, remains largely incomplete. This realization presents a daunting challenge for post-genomic-era scientists in deciphering the biochemical and (patho)physiological roles of these enzymes and their metabolites and metabolic networks. This is further complicated by many recent studies showing a rewiring of normal metabolic networks in disease states to give rise to unique pathophysiological functions of enzymes, metabolites, and metabolic pathways. This review focuses on recent discoveries made using metabolic mapping technologies to uncover novel pathways and metabolite-mediated posttranslational modifications and epigenetic alterations and their impact on physiology and disease. Copyright © 2014 the American Physiological Society.

  14. A limit cycle oscillator model for cycling mood variations of bipolar disorder patients derived from cellular biochemical reaction equations

    NASA Astrophysics Data System (ADS)

    Frank, T. D.

    2013-08-01

    We derive a nonlinear limit cycle model for oscillatory mood variations as observed in patients with cycling bipolar disorder. To this end, we consider two signaling pathways leading to the activation of two enzymes that play a key role for cellular and neural processes. We model pathway cross-talk in terms of an inhibitory impact of the first pathway on the second and an excitatory impact of the second on the first. The model also involves a negative feedback loop (inhibitory self-regulation) for the first pathway and a positive feedback loop (excitatory self-regulation) for the second pathway. We demonstrate that due to the cross-talk the biochemical dynamics is described by an oscillator equation. Under disease-free conditions the oscillatory system exhibits a stable fixed point. The breakdown of the self-inhibition of the first pathway at higher concentration levels is studied by means of a scalar control parameter ξ, where ξ equal to zero refers to intact self-inhibition at all concentration levels. Under certain conditions, stable limit cycle solutions emerge at critical parameter values of ξ larger than zero. These oscillations mimic pathological cycling mood variations that emerge due to a disease-induced bifurcation. Consequently, our modeling analysis supports the notion of bipolar disorder as a dynamical disease. In addition, our study establishes a connection between mechanistic biochemical modeling of bipolar disorder and phenomenological nonlinear oscillator approaches to bipolar disorder suggested in the literature.

  15. Sulfur mobilization for Fe-S cluster assembly by the essential SUF pathway in the Plasmodium falciparum apicoplast and its inhibition.

    PubMed

    Charan, Manish; Singh, Nidhi; Kumar, Bijay; Srivastava, Kumkum; Siddiqi, Mohammad Imran; Habib, Saman

    2014-06-01

    The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

    PubMed Central

    Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

    2013-01-01

    During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

  17. Why is intelligence correlated with semen quality?: Biochemical pathways common to sperm and neuron function and their vulnerability to pleiotropic mutations.

    PubMed

    Pierce, Arand; Miller, Geoffrey; Arden, Rosalind; Gottfredson, Linda S

    2009-09-01

    We recently found positive correlations between human general intelligence and three key indices of semen quality, and hypothesized that these correlations arise through a phenotype-wide 'general fitness factor' reflecting overall mutation load. In this addendum we consider some of the biochemical pathways that may act as targets for pleiotropic mutations that disrupt both neuron function and sperm function in parallel. We focus especially on the inter-related roles of polyunsaturated fatty acids, exocytosis and receptor signaling.

  18. A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

    PubMed Central

    Westerbeck, Jason W.

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics. PMID:26136577

  19. Emerging evidence on the role of the Hippo/YAP pathway in liver physiology and cancer.

    PubMed

    Yimlamai, Dean; Fowl, Brendan H; Camargo, Fernando D

    2015-12-01

    The Hippo pathway and its regulatory target, YAP, has recently emerged as an important biochemical signaling pathway that tightly governs epithelial tissue growth. Initially defined in Drosophilia, this pathway has shown remarkable conservation in vertebrate systems with many components of the Hippo/YAP pathway showing biochemical and functional conservation. The liver is particularly sensitive to changes in Hippo/YAP signaling with rapid increases in liver size becoming manifest on the order of days to weeks after perturbation. The first identified direct targets of Hippo/YAP signaling were pro-proliferative and anti-apoptotic gene programs, but recent work has now implicated this pathway in cell fate choice, stem cell maintenance/renewal, epithelial to mesenchymal transition, and oncogenesis. The mechanisms by which Hippo/YAP signaling is changed endogenously are beginning to come to light as well as how this pathway interacts with other signaling pathways, and important details for designing new therapeutic interventions. This review focuses on the known roles for Hippo/YAP signaling in the liver and promising avenues for future study. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  20. A Cdc48 “Retrochaperone” Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates*

    PubMed Central

    Neal, Sonya; Mak, Raymond; Bennett, Eric J.; Hampton, Randolph

    2017-01-01

    A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical “retrochaperone” for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates. PMID:28077573

  1. A Cdc48 "Retrochaperone" Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates.

    PubMed

    Neal, Sonya; Mak, Raymond; Bennett, Eric J; Hampton, Randolph

    2017-02-24

    A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical "retrochaperone" for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. STEPS: efficient simulation of stochastic reaction-diffusion models in realistic morphologies.

    PubMed

    Hepburn, Iain; Chen, Weiliang; Wils, Stefan; De Schutter, Erik

    2012-05-10

    Models of cellular molecular systems are built from components such as biochemical reactions (including interactions between ligands and membrane-bound proteins), conformational changes and active and passive transport. A discrete, stochastic description of the kinetics is often essential to capture the behavior of the system accurately. Where spatial effects play a prominent role the complex morphology of cells may have to be represented, along with aspects such as chemical localization and diffusion. This high level of detail makes efficiency a particularly important consideration for software that is designed to simulate such systems. We describe STEPS, a stochastic reaction-diffusion simulator developed with an emphasis on simulating biochemical signaling pathways accurately and efficiently. STEPS supports all the above-mentioned features, and well-validated support for SBML allows many existing biochemical models to be imported reliably. Complex boundaries can be represented accurately in externally generated 3D tetrahedral meshes imported by STEPS. The powerful Python interface facilitates model construction and simulation control. STEPS implements the composition and rejection method, a variation of the Gillespie SSA, supporting diffusion between tetrahedral elements within an efficient search and update engine. Additional support for well-mixed conditions and for deterministic model solution is implemented. Solver accuracy is confirmed with an original and extensive validation set consisting of isolated reaction, diffusion and reaction-diffusion systems. Accuracy imposes upper and lower limits on tetrahedron sizes, which are described in detail. By comparing to Smoldyn, we show how the voxel-based approach in STEPS is often faster than particle-based methods, with increasing advantage in larger systems, and by comparing to MesoRD we show the efficiency of the STEPS implementation. STEPS simulates models of cellular reaction-diffusion systems with complex boundaries with high accuracy and high performance in C/C++, controlled by a powerful and user-friendly Python interface. STEPS is free for use and is available at http://steps.sourceforge.net/

  3. Linking gene regulation and the exo-metabolome: A comparative transcriptomics approach to identify genes that impact on the production of volatile aroma compounds in yeast

    PubMed Central

    Rossouw, Debra; Næs, Tormod; Bauer, Florian F

    2008-01-01

    Background 'Omics' tools provide novel opportunities for system-wide analysis of complex cellular functions. Secondary metabolism is an example of a complex network of biochemical pathways, which, although well mapped from a biochemical point of view, is not well understood with regards to its physiological roles and genetic and biochemical regulation. Many of the metabolites produced by this network such as higher alcohols and esters are significant aroma impact compounds in fermentation products, and different yeast strains are known to produce highly divergent aroma profiles. Here, we investigated whether we can predict the impact of specific genes of known or unknown function on this metabolic network by combining whole transcriptome and partial exo-metabolome analysis. Results For this purpose, the gene expression levels of five different industrial wine yeast strains that produce divergent aroma profiles were established at three different time points of alcoholic fermentation in synthetic wine must. A matrix of gene expression data was generated and integrated with the concentrations of volatile aroma compounds measured at the same time points. This relatively unbiased approach to the study of volatile aroma compounds enabled us to identify candidate genes for aroma profile modification. Five of these genes, namely YMR210W, BAT1, AAD10, AAD14 and ACS1 were selected for overexpression in commercial wine yeast, VIN13. Analysis of the data show a statistically significant correlation between the changes in the exo-metabome of the overexpressing strains and the changes that were predicted based on the unbiased alignment of transcriptomic and exo-metabolomic data. Conclusion The data suggest that a comparative transcriptomics and metabolomics approach can be used to identify the metabolic impacts of the expression of individual genes in complex systems, and the amenability of transcriptomic data to direct applications of biotechnological relevance. PMID:18990252

  4. Structure-function analysis of RBP-J-interacting and tubulin-associated (RITA) reveals regions critical for repression of Notch target genes.

    PubMed

    Tabaja, Nassif; Yuan, Zhenyu; Oswald, Franz; Kovall, Rhett A

    2017-06-23

    The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor CSL (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. Although Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog)-interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J·RITA complex formation, and determine the X-ray structure of the RBP-J·RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and isothermal titration calorimetry. Whereas our structural and biophysical studies demonstrate that RITA binds RBP-J similarly to the RAM (RBP-J-associated molecule) domain of Notch, our biochemical and cellular assays suggest that RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. STEPS: efficient simulation of stochastic reaction–diffusion models in realistic morphologies

    PubMed Central

    2012-01-01

    Background Models of cellular molecular systems are built from components such as biochemical reactions (including interactions between ligands and membrane-bound proteins), conformational changes and active and passive transport. A discrete, stochastic description of the kinetics is often essential to capture the behavior of the system accurately. Where spatial effects play a prominent role the complex morphology of cells may have to be represented, along with aspects such as chemical localization and diffusion. This high level of detail makes efficiency a particularly important consideration for software that is designed to simulate such systems. Results We describe STEPS, a stochastic reaction–diffusion simulator developed with an emphasis on simulating biochemical signaling pathways accurately and efficiently. STEPS supports all the above-mentioned features, and well-validated support for SBML allows many existing biochemical models to be imported reliably. Complex boundaries can be represented accurately in externally generated 3D tetrahedral meshes imported by STEPS. The powerful Python interface facilitates model construction and simulation control. STEPS implements the composition and rejection method, a variation of the Gillespie SSA, supporting diffusion between tetrahedral elements within an efficient search and update engine. Additional support for well-mixed conditions and for deterministic model solution is implemented. Solver accuracy is confirmed with an original and extensive validation set consisting of isolated reaction, diffusion and reaction–diffusion systems. Accuracy imposes upper and lower limits on tetrahedron sizes, which are described in detail. By comparing to Smoldyn, we show how the voxel-based approach in STEPS is often faster than particle-based methods, with increasing advantage in larger systems, and by comparing to MesoRD we show the efficiency of the STEPS implementation. Conclusion STEPS simulates models of cellular reaction–diffusion systems with complex boundaries with high accuracy and high performance in C/C++, controlled by a powerful and user-friendly Python interface. STEPS is free for use and is available at http://steps.sourceforge.net/ PMID:22574658

  6. BioWarehouse: a bioinformatics database warehouse toolkit

    PubMed Central

    Lee, Thomas J; Pouliot, Yannick; Wagner, Valerie; Gupta, Priyanka; Stringer-Calvert, David WJ; Tenenbaum, Jessica D; Karp, Peter D

    2006-01-01

    Background This article addresses the problem of interoperation of heterogeneous bioinformatics databases. Results We introduce BioWarehouse, an open source toolkit for constructing bioinformatics database warehouses using the MySQL and Oracle relational database managers. BioWarehouse integrates its component databases into a common representational framework within a single database management system, thus enabling multi-database queries using the Structured Query Language (SQL) but also facilitating a variety of database integration tasks such as comparative analysis and data mining. BioWarehouse currently supports the integration of a pathway-centric set of databases including ENZYME, KEGG, and BioCyc, and in addition the UniProt, GenBank, NCBI Taxonomy, and CMR databases, and the Gene Ontology. Loader tools, written in the C and JAVA languages, parse and load these databases into a relational database schema. The loaders also apply a degree of semantic normalization to their respective source data, decreasing semantic heterogeneity. The schema supports the following bioinformatics datatypes: chemical compounds, biochemical reactions, metabolic pathways, proteins, genes, nucleic acid sequences, features on protein and nucleic-acid sequences, organisms, organism taxonomies, and controlled vocabularies. As an application example, we applied BioWarehouse to determine the fraction of biochemically characterized enzyme activities for which no sequences exist in the public sequence databases. The answer is that no sequence exists for 36% of enzyme activities for which EC numbers have been assigned. These gaps in sequence data significantly limit the accuracy of genome annotation and metabolic pathway prediction, and are a barrier for metabolic engineering. Complex queries of this type provide examples of the value of the data warehousing approach to bioinformatics research. Conclusion BioWarehouse embodies significant progress on the database integration problem for bioinformatics. PMID:16556315

  7. BioWarehouse: a bioinformatics database warehouse toolkit.

    PubMed

    Lee, Thomas J; Pouliot, Yannick; Wagner, Valerie; Gupta, Priyanka; Stringer-Calvert, David W J; Tenenbaum, Jessica D; Karp, Peter D

    2006-03-23

    This article addresses the problem of interoperation of heterogeneous bioinformatics databases. We introduce BioWarehouse, an open source toolkit for constructing bioinformatics database warehouses using the MySQL and Oracle relational database managers. BioWarehouse integrates its component databases into a common representational framework within a single database management system, thus enabling multi-database queries using the Structured Query Language (SQL) but also facilitating a variety of database integration tasks such as comparative analysis and data mining. BioWarehouse currently supports the integration of a pathway-centric set of databases including ENZYME, KEGG, and BioCyc, and in addition the UniProt, GenBank, NCBI Taxonomy, and CMR databases, and the Gene Ontology. Loader tools, written in the C and JAVA languages, parse and load these databases into a relational database schema. The loaders also apply a degree of semantic normalization to their respective source data, decreasing semantic heterogeneity. The schema supports the following bioinformatics datatypes: chemical compounds, biochemical reactions, metabolic pathways, proteins, genes, nucleic acid sequences, features on protein and nucleic-acid sequences, organisms, organism taxonomies, and controlled vocabularies. As an application example, we applied BioWarehouse to determine the fraction of biochemically characterized enzyme activities for which no sequences exist in the public sequence databases. The answer is that no sequence exists for 36% of enzyme activities for which EC numbers have been assigned. These gaps in sequence data significantly limit the accuracy of genome annotation and metabolic pathway prediction, and are a barrier for metabolic engineering. Complex queries of this type provide examples of the value of the data warehousing approach to bioinformatics research. BioWarehouse embodies significant progress on the database integration problem for bioinformatics.

  8. Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery

    PubMed Central

    Bertin, Matthew J.; Kleigrewe, Karin; Leão, Tiago F.; Gerwick, Lena

    2016-01-01

    Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques. PMID:26578313

  9. Reconstructing metabolic flux vectors from extreme pathways: defining the alpha-spectrum.

    PubMed

    Wiback, Sharon J; Mahadevan, Radhakrishnan; Palsson, Bernhard Ø

    2003-10-07

    The move towards genome-scale analysis of cellular functions has necessitated the development of analytical (in silico) methods to understand such large and complex biochemical reaction networks. One such method is extreme pathway analysis that uses stoichiometry and thermodynamic irreversibly to define mathematically unique, systemic metabolic pathways. These extreme pathways form the edges of a high-dimensional convex cone in the flux space that contains all the attainable steady state solutions, or flux distributions, for the metabolic network. By definition, any steady state flux distribution can be described as a nonnegative linear combination of the extreme pathways. To date, much effort has been focused on calculating, defining, and understanding these extreme pathways. However, little work has been performed to determine how these extreme pathways contribute to a given steady state flux distribution. This study represents an initial effort aimed at defining how physiological steady state solutions can be reconstructed from a network's extreme pathways. In general, there is not a unique set of nonnegative weightings on the extreme pathways that produce a given steady state flux distribution but rather a range of possible values. This range can be determined using linear optimization to maximize and minimize the weightings of a particular extreme pathway in the reconstruction, resulting in what we have termed the alpha-spectrum. The alpha-spectrum defines which extreme pathways can and cannot be included in the reconstruction of a given steady state flux distribution and to what extent they individually contribute to the reconstruction. It is shown that accounting for transcriptional regulatory constraints can considerably shrink the alpha-spectrum. The alpha-spectrum is computed and interpreted for two cases; first, optimal states of a skeleton representation of core metabolism that include transcriptional regulation, and second for human red blood cell metabolism under various physiological, non-optimal conditions.

  10. New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide

    PubMed Central

    2014-01-01

    Background In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD+de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD+ salvage pathway from nicotinamide. Results Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Conclusions Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD+ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD+ salvage pathway might be significant in some bacteria lacking NAD+de novo and NAD+ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD+. However, this speculation needs to be experimentally tested. PMID:24506841

  11. The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

    PubMed

    Pey, Angel L; Majtan, Tomas; Kraus, Jan P

    2014-09-01

    Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse

    PubMed Central

    Chao, Yu-Hsin; Giagtzoglou, Nikolaos; Putluri, Nagireddy; Coarfa, Cristian; Donti, Taraka; Faust, Joseph E.; McNew, James A.; Sardiello, Marco; Baes, Myriam; Bellen, Hugo J.

    2017-01-01

    Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD. PMID:28640802

  13. New insight on the S100A1-STIP1 complex highlights the important relationship between allostery and entropy in protein function.

    PubMed

    Nucci, Nathaniel V

    2017-08-17

    Calcium signaling serves as a nexus of many vital cellular processes. Of particular importance is the role the calcium signaling plays in the prevention of protein misfolding, and the S100 family of calcium-binding proteins is a key player in this pathway. While the S100 proteins carry out a range of roles, the interaction of S100A1 and the stress-inducible phosphoprotein 1 (STIP1) has been shown to be particularly important. A recent study by Maciejewski et al. in Biochemical Journal ( Biochemical Journal (2017) 474 , 1853-1866) revealed new insights into the nature of the S100A1-STIP1 interaction. Not only did the present paper indicate the stoichiometry of binding for this interaction (three S100A1 dimers : one STIP1), it also demonstrated that the binding interaction is highly co-operative and that each S100A1-STIP1-binding interaction is entropically driven. The findings presented raise important new questions regarding the relationship between entropy and allostery in protein function. Recently, the dynamical underpinnings of allostery in protein function have become a topic of increased interest. A broad range of investigations have demonstrated that allostery can be mediated by entropic processes such as changes in the flexibility of the protein backbone and in the range of motions explored by side chains. The S100A1-STIP1 complex as described by Maciejewski et al. suggests a new system in which an allosteric-binding interaction driven by entropic processes may be systematically dissected in the future. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  14. Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue*

    PubMed Central

    MacDonald, Matthew L.; Ciccimaro, Eugene; Prakash, Amol; Banerjee, Anamika; Seeholzer, Steven H.; Blair, Ian A.; Hahn, Chang-Gyu

    2012-01-01

    Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. PMID:22942359

  15. Adverse Outcome Pathways: From Definition to Application

    EPA Science Inventory

    A challenge for both human health and ecological toxicologists is the transparent application of mechanistic (e.g., molecular, biochemical, histological) data to risk assessments. The adverse outcome pathway (AOP) is a conceptual framework designed to meet this need. Specifical...

  16. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  17. Drosophila as a model system to study autophagy.

    PubMed

    Zirin, Jonathan; Perrimon, Norbert

    2010-12-01

    Originally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context.

  18. Peroxisome protein import: a complex journey.

    PubMed

    Baker, Alison; Lanyon-Hogg, Thomas; Warriner, Stuart L

    2016-06-15

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. © 2016 The Author(s).

  19. ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

    PubMed

    Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

    2017-10-05

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  20. Explorations into Chemical Reactions and Biochemical Pathways.

    PubMed

    Gasteiger, Johann

    2016-12-01

    A brief overview of the work in the research group of the present author on extracting knowledge from chemical reaction data is presented. Methods have been developed to calculate physicochemical effects at the reaction site. It is shown that these physicochemical effects can quite favourably be used to derive equations for the calculation of data on gas phase reactions and on reactions in solution such as aqueous acidity of alcohols or carboxylic acids or the hydrolysis of amides. Furthermore, it is shown that these physicochemical effects are quite effective for assigning reactions into reaction classes that correspond to chemical knowledge. Biochemical reactions constitute a particularly interesting and challenging task for increasing our understanding of living species. The BioPath.Database is a rich source of information on biochemical reactions and has been used for a variety of applications of chemical, biological, or medicinal interests. Thus, it was shown that biochemical reactions can be assigned by the physicochemical effects into classes that correspond to the classification of enzymes by the EC numbers. Furthermore, 3D models of reaction intermediates can be used for searching for novel enzyme inhibitors. It was shown in a combined application of chemoinformatics and bioinformatics that essential pathways of diseases can be uncovered. Furthermore, a study showed that bacterial flavor-forming pathways can be discovered. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Diabetic Neuropathy: Mechanisms to Management

    PubMed Central

    Edwards, James L.; Vincent, Andrea; Cheng, Thomas; Feldman, Eva L.

    2014-01-01

    Neuropathy is the most common and debilitating complication of diabetes and results in pain, decreased motility, and amputation. Diabetic neuropathy encompasses a variety of forms whose impact ranges from discomfort to death. Hyperglycemia induces oxidative stress in diabetic neurons and results in activation of multiple biochemical pathways. These activated pathways are a major source of damage and are potential therapeutic targets in diabetic neuropathy. Though therapies are available to alleviate the symptoms of diabetic neuropathy, few options are available to eliminate the root causes. The immense physical, psychological, and economic cost of diabetic neuropathy underscores the need for causally targeted therapies. This review covers the pathology, epidemiology, biochemical pathways, and prevention of diabetic neuropathy, as well as discusses current symptomatic and causal therapies and novel approaches to identify therapeutic targets. PMID:18616962

  2. Mitochondrial Calcium Transport in Trypanosomes

    PubMed Central

    Docampo, Roberto; Vercesi, Anibal E.; Huang, Guozhong

    2014-01-01

    The biochemical peculiarities of trypanosomes were fundamental for the recent molecular identification of the long-sought channel involved in mitochondrial Ca2+ uptake, the mitochondrial Ca2+ uniporter or MCU. This discovery led to the finding of numerous regulators of the channel, which form a high molecular weight complex with MCU. Some of these regulators have been bioinformatically identified in trypanosomes, which are the first eukaryotic organisms described for which MCU is essential. In trypanosomes MCU is important for buffering cytosolic Ca2+ changes and for activation of the bioenergetics of the cells. Future work on this pathway in trypanosomes promises further insight into the biology of these fascinating eukaryotes, as well as the potential for novel target discovery. PMID:25218432

  3. Cellular damage in bacterial meningitis: an interplay of bacterial and host driven toxicity.

    PubMed

    Weber, Joerg R; Tuomanen, Elaine I

    2007-03-01

    Bacterial meningitis is still an important infectious disease causing death and disability. Invasive bacterial infections of the CNS generate some of the most powerful inflammatory responses known in medicine. Although the components of bacterial cell surfaces are now chemically defined in exquisite detail and the interaction with several receptor pathways has been discovered, it is only very recently that studies combining these advanced biochemical and cell biological tools have been done. Additional to the immunological response direct bacterial toxicity has been identified as an important contributor to neuronal damage. A detailed understanding of the complex interaction of bacterial toxicity and host response may generate opportunities for innovative and specific neuroprotective therapies.

  4. Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts

    PubMed Central

    Misra, Rajeev

    2012-01-01

    In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts. PMID:27335668

  5. Signaling pathways that regulate life and cell death: evolution of apoptosis in the context of self-defense.

    PubMed

    Muñoz-Pinedo, Cristina

    2012-01-01

    Programmed Cell Death is essential for the life cycle of many organisms. Cell death in multicellular organisms can occur as a consequence of massive damage (necrosis) or in a controlled form, through engagement of diverse biochemical programs. The best well known form of programmed cell death is apoptosis. Apoptosis occurs in animals as a consequence of a variety of stimuli including stress and social signals and it plays essential roles in morphogenesis and immune defense. The machinery of apoptosis is well conserved among animals and it is composed of caspases (the proteases which execute cell death), adapter proteins (caspase activators), Bcl-2 family proteins and Inhibitor of Apoptosis Proteins (IAPs). We will describe in this chapter the main apoptotic pathways in animals: the extrinsic (death receptor-mediated), the intrinsic/mitochondrial and the Granzyme B pathway. Other forms of non-apoptotic Programmed Cell Death which occur in animals will also be discussed. We will summarize the current knowledge about apoptotic-like and other forms of cell death in other organisms such as plants and protists.Additionally, we will discuss the hypothesis that apoptosis originated as part of a host defense mechanism. We will explore the similarities between the protein complexes which mediate apoptosis (apoptosomes) and complexes involved in immunity: inflammasomes. Additional functions of apoptotic proteins related to immune function will be summarized, in an effort to explore the evolutionary origins of cell death.

  6. Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

    PubMed Central

    Upadhyay, Srijana; Xu, Xinping

    2016-01-01

    ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

  7. The Modular Organization of Protein Interactions in Escherichia coli

    PubMed Central

    Peregrín-Alvarez, José M.; Xiong, Xuejian; Su, Chong; Parkinson, John

    2009-01-01

    Escherichia coli serves as an excellent model for the study of fundamental cellular processes such as metabolism, signalling and gene expression. Understanding the function and organization of proteins within these processes is an important step towards a ‘systems’ view of E. coli. Integrating experimental and computational interaction data, we present a reliable network of 3,989 functional interactions between 1,941 E. coli proteins (∼45% of its proteome). These were combined with a recently generated set of 3,888 high-quality physical interactions between 918 proteins and clustered to reveal 316 discrete modules. In addition to known protein complexes (e.g., RNA and DNA polymerases), we identified modules that represent biochemical pathways (e.g., nitrate regulation and cell wall biosynthesis) as well as batteries of functionally and evolutionarily related processes. To aid the interpretation of modular relationships, several case examples are presented, including both well characterized and novel biochemical systems. Together these data provide a global view of the modular organization of the E. coli proteome and yield unique insights into structural and evolutionary relationships in bacterial networks. PMID:19798435

  8. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  9. Cellular and molecular mechanisms for the bone response to mechanical loading

    NASA Technical Reports Server (NTRS)

    Bloomfield, S. A.

    2001-01-01

    To define the cellular and molecular mechanisms for the osteogenic response of bone to increased loading, several key steps must be defined: sensing of the mechanical signal by cells in bone, transduction of the mechanical signal to a biochemical one, and transmission of that biochemical signal to effector cells. Osteocytes are likely to serve as sensors of loading, probably via interstitial fluid flow produced during loading. Evidence is presented for the role of integrins, the cell's actin cytoskeleton, G proteins, and various intracellular signaling pathways in transducing that mechanical signal to a biochemical one. Nitric oxide, prostaglandins, and insulin-like growth factors all play important roles in these pathways. There is growing evidence for modulation of these mechanotransduction steps by endocrine factors, particularly parathyroid hormone and estrogen. The efficiency of this process is also impaired in the aged animal, yet what remains undefined is at what step mechanotransduction is affected.

  10. Magnetic resonance microscopy: concepts, challenges, and state-of-the-art.

    PubMed

    Gimi, Barjor

    2006-01-01

    Recent strides in targeted therapy and regenerative medicine have created a need to identify molecules and metabolic pathways implicated in a disease and its treatment. These molecules and pathways must be discerned at the cellular level to meaningfully reveal the biochemical underpinnings of the disease and to identify key molecular targets for therapy. Magnetic resonance (MR) techniques are well suited for molecular and functional imaging because of their noninvasive nature and their versatility in extracting physiological, biochemical, and functional information over time. However, MR is an insensitive technique; MR microscopy seeks to increase detection sensitivity, thereby localizing biochemical and functional information at the level of single cells or small cellular clusters. Here, we discuss some of the challenges facing MR microscopy and the technical and phenomenological strategies used to overcome these challenges. Some of the applications of MR microscopy are highlighted in this chapter.

  11. Teaching arrangements of carbohydrate metabolism in biochemistry curriculum in Peking University Health Science Center.

    PubMed

    Chen, Hao; Ni, Ju-Hua

    2013-01-01

    Biochemistry occupies a unique place in the medical school curricula, but the teaching of biochemistry presents certain challenges. One of these challenges is facilitating students' interest in and mastery of metabolism. The many pathways and modes of regulation can be overwhelming for students to learn and difficult for professors to teach in an engaging manner. The first chapter of the metabolism section in current Chinese biochemistry textbooks covers carbohydrate metabolism. Medical students usually complain about the difficulty of this subject. Here we discuss how to facilitate learning by rearranging the subjects in this introductory chapter of biochemical metabolism and to lay a solid foundation for future study. The strategy involves reorganizing the order in which subjects are taught from simple to complex and from short to long metabolic pathways. Most students taking the curriculum consider that the strategy engages their learning interests in biochemistry and enhances their learning outcomes. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  12. Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

    PubMed Central

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

  13. Multiscale Modelling of Cancer Progression and Treatment Control: The Role of Intracellular Heterogeneities in Chemotherapy Treatment

    NASA Astrophysics Data System (ADS)

    Chaplain, Mark A. J.; Powathil, Gibin G.

    Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.

  14. Multiscale Modelling of Cancer Progression and Treatment Control: The Role of Intracellular Heterogeneities in Chemotherapy Treatment

    NASA Astrophysics Data System (ADS)

    Chaplain, Mark A. J.; Powathil, Gibin G.

    2015-04-01

    Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.

  15. Integrating computational methods to retrofit enzymes to synthetic pathways.

    PubMed

    Brunk, Elizabeth; Neri, Marilisa; Tavernelli, Ivano; Hatzimanikatis, Vassily; Rothlisberger, Ursula

    2012-02-01

    Microbial production of desired compounds provides an efficient framework for the development of renewable energy resources. To be competitive to traditional chemistry, one requirement is to utilize the full capacity of the microorganism to produce target compounds with high yields and turnover rates. We use integrated computational methods to generate and quantify the performance of novel biosynthetic routes that contain highly optimized catalysts. Engineering a novel reaction pathway entails addressing feasibility on multiple levels, which involves handling the complexity of large-scale biochemical networks while respecting the critical chemical phenomena at the atomistic scale. To pursue this multi-layer challenge, our strategy merges knowledge-based metabolic engineering methods with computational chemistry methods. By bridging multiple disciplines, we provide an integral computational framework that could accelerate the discovery and implementation of novel biosynthetic production routes. Using this approach, we have identified and optimized a novel biosynthetic route for the production of 3HP from pyruvate. Copyright © 2011 Wiley Periodicals, Inc.

  16. Metabolic engineering of microorganisms for the synthesis of plant natural products.

    PubMed

    Marienhagen, Jan; Bott, Michael

    2013-01-20

    Of more than 200,000 plant natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance and environmental, seasonal as well as regional variation, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most plant natural products. With advances in DNA sequencing and recombinant DNA technology many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways in suitable microorganisms. This approach offers promise to provide sufficient quantities of the desired plant natural products from inexpensive renewable resources. This review covers recent advancements in the metabolic engineering of microorganisms for the production of plant natural products such as isoprenoids, phenylpropanoids and alkaloids, and highlights general approaches and strategies to gain access to the rich biochemical diversity of plants by employing the biosynthetic power of microorganisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production.

    PubMed

    Streit, W R; Entcheva, P

    2003-03-01

    Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10-30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.

  18. Stable-Carbon Isotopic Composition of Maple Sap and Foliage 1

    PubMed Central

    Leavitt, Steven W.; Long, Austin

    1985-01-01

    The 13C/12C ratios of Acer grandidentatum sap sugar collected during the dormant period are compared to those of buds, leaves, and wood developed over the following growing season. As the primary carbon source for cellulose manufacture at initiation of annual growth in deciduous trees, sap sucrose would be expected to have an isotopic composition similar to first-formed cellulose. Although constancy in concentration and 13C/12C ratios of the maple sap sugar suggests any gains or losses (e.g. to maintenance metabolism) do not appreciably alter composition, the 13C/12C ratios of cellulose of the enlarging buds in the spring are quite distinct from those of the sap sugar, seemingly precluding a simple direct biochemical pathway of sap sucrose→glucose→cellulose in favor of a more complex pathway with greater likelihood of isotopic fractionation. The 13C/12C ratios of the leaves and in the growth ring were initially similar to the sap sugar but decreased steadily over the growing season. PMID:16664259

  19. Stable-carbon isotopic composition of maple sap and foliage.

    PubMed

    Leavitt, S W; Long, A

    1985-06-01

    The (13)C/(12)C ratios of Acer grandidentatum sap sugar collected during the dormant period are compared to those of buds, leaves, and wood developed over the following growing season. As the primary carbon source for cellulose manufacture at initiation of annual growth in deciduous trees, sap sucrose would be expected to have an isotopic composition similar to first-formed cellulose. Although constancy in concentration and (13)C/(12)C ratios of the maple sap sugar suggests any gains or losses (e.g. to maintenance metabolism) do not appreciably alter composition, the (13)C/(12)C ratios of cellulose of the enlarging buds in the spring are quite distinct from those of the sap sugar, seemingly precluding a simple direct biochemical pathway of sap sucrose-->glucose-->cellulose in favor of a more complex pathway with greater likelihood of isotopic fractionation. The (13)C/(12)C ratios of the leaves and in the growth ring were initially similar to the sap sugar but decreased steadily over the growing season.

  20. Metabolic engineering approaches for production of biochemicals in food and medicinal plants.

    PubMed

    Wilson, Sarah A; Roberts, Susan C

    2014-04-01

    Historically, plants are a vital source of nutrients and pharmaceuticals. Recent advances in metabolic engineering have made it possible to not only increase the concentration of desired compounds, but also introduce novel biosynthetic pathways to a variety of species, allowing for enhanced nutritional or commercial value. To improve metabolic engineering capabilities, new transformation techniques have been developed to allow for gene specific silencing strategies or stacking of multiple genes within the same region of the chromosome. The 'omics' era has provided a new resource for elucidation of uncharacterized biosynthetic pathways, enabling novel metabolic engineering approaches. These resources are now allowing for advanced metabolic engineering of plant production systems, as well as the synthesis of increasingly complex products in engineered microbial hosts. The status of current metabolic engineering efforts is highlighted for the in vitro production of paclitaxel and the in vivo production of β-carotene in Golden Rice and other food crops. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Decomposition of complex microbial behaviors into resource-based stress responses

    PubMed Central

    Carlson, Ross P.

    2009-01-01

    Motivation: Highly redundant metabolic networks and experimental data from cultures likely adapting simultaneously to multiple stresses can complicate the analysis of cellular behaviors. It is proposed that the explicit consideration of these factors is critical to understanding the competitive basis of microbial strategies. Results: Wide ranging, seemingly unrelated Escherichia coli physiological fluxes can be simply and accurately described as linear combinations of a few ecologically relevant stress adaptations. These strategies were identified by decomposing the central metabolism of E.coli into elementary modes (mathematically defined biochemical pathways) and assessing the resource investment cost–benefit properties for each pathway. The approach capitalizes on the inherent tradeoffs related to investing finite resources like nitrogen into different pathway enzymes when the pathways have varying metabolic efficiencies. The subset of ecologically competitive pathways represented 0.02% of the total permissible pathways. The biological relevance of the assembled strategies was tested against 10 000 randomly constructed pathway subsets. None of the randomly assembled collections were able to describe all of the considered experimental data as accurately as the cost-based subset. The results suggest these metabolic strategies are biologically significant. The current descriptions were compared with linear programming (LP)-based flux descriptions using the Euclidean distance metric. The current study's pathway subset described the experimental fluxes with better accuracy than the LP results without having to test multiple objective functions or constraints and while providing additional ecological insight into microbial behavior. The assembled pathways seem to represent a generalized set of strategies that can describe a wide range of microbial responses and hint at evolutionary processes where a handful of successful metabolic strategies are utilized simultaneously in different combinations to adapt to diverse conditions. Contact: rossc@biofilms.montana.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19008248

  2. Therapeutic modulation of cerebral L-lysine metabolism in a mouse model for glutaric aciduria type I.

    PubMed

    Sauer, Sven W; Opp, Silvana; Hoffmann, Georg F; Koeller, David M; Okun, Jürgen G; Kölker, Stefan

    2011-01-01

    Glutaric aciduria type I, an inherited deficiency of glutaryl-coenzyme A dehydrogenase localized in the final common catabolic pathway of L-lysine, L-hydroxylysine and L-tryptophan, leads to accumulation of neurotoxic glutaric and 3-hydroxyglutaric acid, as well as non-toxic glutarylcarnitine. Most untreated patients develop irreversible brain damage during infancy that can be prevented in the majority of cases if metabolic treatment with a low L-lysine diet and L-carnitine supplementation is started in the newborn period. The biochemical effect of this treatment remains uncertain, since cerebral concentrations of neurotoxic metabolites can only be determined by invasive techniques. Therefore, we studied the biochemical effect and mechanism of metabolic treatment in glutaryl-coenzyme A dehydrogenase-deficient mice, an animal model with complete loss of glutaryl-coenzyme A dehydrogenase activity, focusing on the tissue-specific changes of neurotoxic metabolites and key enzymes of L-lysine metabolism. Here, we demonstrate that low L-lysine diet, but not L-carnitine supplementation, lowered the concentration of glutaric acid in brain, liver, kidney and serum. L-carnitine supplementation restored the free L-carnitine pool and enhanced the formation of glutarylcarnitine. The effect of low L-lysine diet was amplified by add-on therapy with L-arginine, which we propose to result from competition with L-lysine at system y(+) of the blood-brain barrier and the mitochondrial L-ornithine carriers. L-lysine can be catabolized in the mitochondrial saccharopine or the peroxisomal pipecolate pathway. We detected high activity of mitochondrial 2-aminoadipate semialdehyde synthase, the rate-limiting enzyme of the saccharopine pathway, in the liver, whereas it was absent in the brain. Since we found activity of the subsequent enzymes of L-lysine oxidation, 2-aminoadipate semialdehyde dehydrogenase, 2-aminoadipate aminotransferase and 2-oxoglutarate dehydrogenase complex as well as peroxisomal pipecolic acid oxidase in brain tissue, we postulate that the pipecolate pathway is the major route of L-lysine degradation in the brain and the saccharopine pathway is the major route in the liver. Interestingly, treatment with clofibrate decreased cerebral and hepatic concentrations of glutaric acid in glutaryl-coenzyme A dehydrogenase-deficient mice. This finding opens new therapeutic perspectives such as pharmacological stimulation of alternative L-lysine oxidation in peroxisomes. In conclusion, this study gives insight into the discrepancies between cerebral and hepatic L-lysine metabolism, provides for the first time a biochemical proof of principle for metabolic treatment in glutaric aciduria type I and suggests that further optimization of treatment could be achieved by exploitation of competition between L-lysine and L-arginine at physiological barriers and enhancement of peroxisomal L-lysine oxidation and glutaric acid breakdown.

  3. Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae.

    PubMed

    Li, Mingji; Borodina, Irina

    2015-02-01

    Synthetic biology and metabolic engineering enable generation of novel cell factories that efficiently convert renewable feedstocks into biofuels, bulk, and fine chemicals, thus creating the basis for biosustainable economy independent on fossil resources. While over a hundred proof-of-concept chemicals have been made in yeast, only a very small fraction of those has reached commercial-scale production so far. The limiting factor is the high research cost associated with the development of a robust cell factory that can produce the desired chemical at high titer, rate, and yield. Synthetic biology has the potential to bring down this cost by improving our ability to predictably engineer biological systems. This review highlights synthetic biology applications for design, assembly, and optimization of non-native biochemical pathways in baker's yeast Saccharomyces cerevisiae We describe computational tools for the prediction of biochemical pathways, molecular biology methods for assembly of DNA parts into pathways, and for introducing the pathways into the host, and finally approaches for optimizing performance of the introduced pathways. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  4. Murine models of atrophy, cachexia, and sarcopenia in skeletal muscle

    PubMed Central

    Romanick, Mark; Brown-Borg, Holly M.

    2013-01-01

    With the extension of life span over the past several decades, the age-related loss of muscle mass and strength that characterizes sarcopenia is becoming more evident and thus, has a more significant impact on society. To determine ways to intervene and delay, or even arrest the physical frailty and dependence that accompany sarcopenia, it is necessary to identify those biochemical pathways that define this process. Animal models that mimic one or more of the physiological pathways involved with this phenomenon are very beneficial in providing an understanding of the cellular processes at work in sarcopenia. The ability to influence pathways through genetic manipulation gives insight into cellular responses and their impact on the physical expression of sarcopenia. This review evaluates several murine models that have the potential to elucidate biochemical processes integral to sarcopenia. Identifying animal models that reflect sarcopenia or its component pathways will enable researchers to better understand those pathways that contribute to age-related skeletal muscle mass loss, and in turn, develop interventions that will prevent, retard, arrest, or reverse this phenomenon. PMID:23523469

  5. Disruption of Abscisic Acid Signaling Constitutively Activates Arabidopsis Resistance to the Necrotrophic Fungus Plectosphaerella cucumerina1[W

    PubMed Central

    Sánchez-Vallet, Andrea; López, Gemma; Ramos, Brisa; Delgado-Cerezo, Magdalena; Riviere, Marie-Pierre; Llorente, Francisco; Fernández, Paula Virginia; Miedes, Eva; Estevez, José Manuel; Grant, Murray; Molina, Antonio

    2012-01-01

    Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi. PMID:23037505

  6. The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans.

    PubMed

    Alam, Md Ashiqul; Kamlangdee, Niyom; Kelly, Joan M

    2017-08-01

    Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.

  7. Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

    PubMed Central

    Kozak, Barbara U.; van Rossum, Harmen M.; Luttik, Marijke A. H.; Akeroyd, Michiel; Benjamin, Kirsten R.; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T.

    2014-01-01

    ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. PMID:25336454

  8. Analysis of selected volatile organic compounds in split and nonsplit swiss cheese samples using selected-ion flow tube mass spectrometry (SIFT-MS).

    PubMed

    Castada, Hardy Z; Wick, Cheryl; Taylor, Kaitlyn; Harper, W James

    2014-04-01

    Splits/cracks are recurring product defects that negatively affect the Swiss cheese industry. Investigations to understand the biophysicochemical aspects of these defects, and thus determine preventive measures against their occurrence, are underway. In this study, selected-ion, flow tube mass spectrometry was employed to determine the volatile organic compound (VOC) profiles present in the headspace of split compared with nonsplit cheeses. Two sampling methodologies were employed: split compared with nonsplit cheese vat pair blocks; and comparison of blind, eye, and split segments within cheese blocks. The variability in VOC profiles was examined to evaluate the potential biochemical pathway chemistry differences within and between cheese samples. VOC profile inhomogeneity was most evident in cheeses between factories. Evaluation of biochemical pathways leading to the formation of key VOCs differentiating the split from the blind and eye segments within factories indicated release of additional carbon dioxide by-product. These results suggest a factory-dependent cause of split formation that could develop from varied fermentation pathways in the blind, eye, and split areas within a cheese block. The variability of VOC profiles within and between factories exhibit varied biochemical fermentation pathways that could conceivably be traced back in the making process to identify parameters responsible for split defect. © 2014 Institute of Food Technologists®

  9. Phototropism: growing towards an understanding of plant movement.

    PubMed

    Liscum, Emmanuel; Askinosie, Scott K; Leuchtman, Daniel L; Morrow, Johanna; Willenburg, Kyle T; Coats, Diana Roberts

    2014-01-01

    Phototropism, or the differential cell elongation exhibited by a plant organ in response to directional blue light, provides the plant with a means to optimize photosynthetic light capture in the aerial portion and water and nutrient acquisition in the roots. Tremendous advances have been made in our understanding of the molecular, biochemical, and cellular bases of phototropism in recent years. Six photoreceptors and their associated signaling pathways have been linked to phototropic responses under various conditions. Primary detection of directional light occurs at the plasma membrane, whereas secondary modulatory photoreception occurs in the cytoplasm and nucleus. Intracellular responses to light cues are processed to regulate cell-to-cell movement of auxin to allow establishment of a trans-organ gradient of the hormone. Photosignaling also impinges on the transcriptional regulation response established as a result of changes in local auxin concentrations. Three additional phytohormone signaling pathways have also been shown to influence phototropic responsiveness, and these pathways are influenced by the photoreceptor signaling as well. Here, we will discuss this complex dance of intra- and intercellular responses that are regulated by these many systems to give rise to a rapid and robust adaptation response observed as organ bending.

  10. Phototropism: Growing towards an Understanding of Plant Movement[OPEN

    PubMed Central

    Liscum, Emmanuel; Askinosie, Scott K.; Leuchtman, Daniel L.; Morrow, Johanna; Willenburg, Kyle T.; Coats, Diana Roberts

    2014-01-01

    Phototropism, or the differential cell elongation exhibited by a plant organ in response to directional blue light, provides the plant with a means to optimize photosynthetic light capture in the aerial portion and water and nutrient acquisition in the roots. Tremendous advances have been made in our understanding of the molecular, biochemical, and cellular bases of phototropism in recent years. Six photoreceptors and their associated signaling pathways have been linked to phototropic responses under various conditions. Primary detection of directional light occurs at the plasma membrane, whereas secondary modulatory photoreception occurs in the cytoplasm and nucleus. Intracellular responses to light cues are processed to regulate cell-to-cell movement of auxin to allow establishment of a trans-organ gradient of the hormone. Photosignaling also impinges on the transcriptional regulation response established as a result of changes in local auxin concentrations. Three additional phytohormone signaling pathways have also been shown to influence phototropic responsiveness, and these pathways are influenced by the photoreceptor signaling as well. Here, we will discuss this complex dance of intra- and intercellular responses that are regulated by these many systems to give rise to a rapid and robust adaptation response observed as organ bending. PMID:24481074

  11. Systems Biology Graphical Notation: Process Description language Level 1 Version 1.3.

    PubMed

    Moodie, Stuart; Le Novère, Nicolas; Demir, Emek; Mi, Huaiyu; Villéger, Alice

    2015-09-04

    The Systems Biological Graphical Notation (SBGN) is an international community effort for standardized graphical representations of biological pathways and networks. The goal of SBGN is to provide unambiguous pathway and network maps for readers with different scientific backgrounds as well as to support efficient and accurate exchange of biological knowledge between different research communities, industry, and other players in systems biology. Three SBGN languages, Process Description (PD), Entity Relationship (ER) and Activity Flow (AF), allow for the representation of different aspects of biological and biochemical systems at different levels of detail. The SBGN Process Description language represents biological entities and processes between these entities within a network. SBGN PD focuses on the mechanistic description and temporal dependencies of biological interactions and transformations. The nodes (elements) are split into entity nodes describing, e.g., metabolites, proteins, genes and complexes, and process nodes describing, e.g., reactions and associations. The edges (connections) provide descriptions of relationships (or influences) between the nodes, such as consumption, production, stimulation and inhibition. Among all three languages of SBGN, PD is the closest to metabolic and regulatory pathways in biological literature and textbooks, but its well-defined semantics offer a superior precision in expressing biological knowledge.

  12. Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics.

    PubMed

    Sridharan, Gautham Vivek; Bruinsma, Bote Gosse; Bale, Shyam Sundhar; Swaminathan, Anandh; Saeidi, Nima; Yarmush, Martin L; Uygun, Korkut

    2017-11-13

    Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses.

  13. Systems biology: a new tool for farm animal science.

    PubMed

    Hollung, Kristin; Timperio, Anna M; Olivan, Mamen; Kemp, Caroline; Coto-Montes, Ana; Sierra, Veronica; Zolla, Lello

    2014-03-01

    It is rapidly emerging that the tender meat phenotype is affected by an enormous amount of variables, not only tied to genetics (livestock breeding selection), but also to extrinsic factors, such as feeding conditions, physical activity, rearing environment, administration of hormonal growth promotants, pre-slaughter handling and stress. Proteomics has been widely accepted by meat scientists over the last years and is now commonly used to shed light on the postmortem processes involved in meat tenderization. This review discusses the latest findings with the use of proteomics and systems biology to study the different biochemical pathways postmortem aiming at understanding the concerted action of different molecular mechanisms responsible for meat quality. The conversion of muscle to meat postmortem can be described as a sequence of events involving molecular pathways controlled by a complex interplay of many factors. Among the different pathways emerging are the influence of apoptosis and lately also the role of autophagy in muscle postmortem development. This review thus, focus on how systems-wide integrated investigations (metabolomics, transcriptomics, interactomics, phosphoproteomics, mathematical modeling), which have emerged as complementary tools to proteomics, have helped establishing a few milestones in our understanding of the events leading from muscle to meat conversion.

  14. Selenium uptake, translocation, assimilation and metabolic fate in plants.

    PubMed

    Sors, T G; Ellis, D R; Salt, D E

    2005-12-01

    The chemical and physical resemblance between selenium (Se) and sulfur (S) establishes that both these elements share common metabolic pathways in plants. The presence of isologous Se and S compounds indicates that these elements compete in biochemical processes that affect uptake, translocation and assimilation throughout plant development. Yet, minor but crucial differences in reactivity and other metabolic interactions infer that some biochemical processes involving Se may be excluded from those relating to S. This review examines the current understanding of physiological and biochemical relationships between S and Se metabolism by highlighting their similarities and differences in relation to uptake, transport and assimilation pathways as observed in Se hyperaccumulator and non-accumulator plant species. The exploitation of genetic resources used in bioengineering strategies of plants is illuminating the function of sulfate transporters and key enzymes of the S assimilatory pathway in relation to Se accumulation and final metabolic fate. These strategies are providing the basic framework by which to resolve questions relating to the essentiality of Se in plants and the mechanisms utilized by Se hyperaccumulators to circumvent toxicity. In addition, such approaches may assist in the future application of genetically engineered Se accumulating plants for environmental renewal and human health objectives.

  15. Cellular Signaling Networks Function as Generalized Wiener-Kolmogorov Filters to Suppress Noise

    NASA Astrophysics Data System (ADS)

    Hinczewski, Michael; Thirumalai, D.

    2014-10-01

    Cellular signaling involves the transmission of environmental information through cascades of stochastic biochemical reactions, inevitably introducing noise that compromises signal fidelity. Each stage of the cascade often takes the form of a kinase-phosphatase push-pull network, a basic unit of signaling pathways whose malfunction is linked with a host of cancers. We show that this ubiquitous enzymatic network motif effectively behaves as a Wiener-Kolmogorov optimal noise filter. Using concepts from umbral calculus, we generalize the linear Wiener-Kolmogorov theory, originally introduced in the context of communication and control engineering, to take nonlinear signal transduction and discrete molecule populations into account. This allows us to derive rigorous constraints for efficient noise reduction in this biochemical system. Our mathematical formalism yields bounds on filter performance in cases important to cellular function—such as ultrasensitive response to stimuli. We highlight features of the system relevant for optimizing filter efficiency, encoded in a single, measurable, dimensionless parameter. Our theory, which describes noise control in a large class of signal transduction networks, is also useful both for the design of synthetic biochemical signaling pathways and the manipulation of pathways through experimental probes such as oscillatory input.

  16. Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes.

    PubMed

    Huang, Ruiqi; O'Donnell, Andrew J; Barboline, Jessica J; Barkman, Todd J

    2016-09-20

    Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

  17. Anaerobic Degradation of Benzene and Polycyclic Aromatic Hydrocarbons.

    PubMed

    Meckenstock, Rainer U; Boll, Matthias; Mouttaki, Housna; Koelschbach, Janina S; Cunha Tarouco, Paola; Weyrauch, Philip; Dong, Xiyang; Himmelberg, Anne M

    2016-01-01

    Aromatic hydrocarbons such as benzene and polycyclic aromatic hydrocarbons (PAHs) are very slowly degraded without molecular oxygen. Here, we review the recent advances in the elucidation of the first known degradation pathways of these environmental hazards. Anaerobic degradation of benzene and PAHs has been successfully documented in the environment by metabolite analysis, compound-specific isotope analysis and microcosm studies. Subsequently, also enrichments and pure cultures were obtained that anaerobically degrade benzene, naphthalene or methylnaphthalene, and even phenanthrene, the largest PAH currently known to be degradable under anoxic conditions. Although such cultures grow very slowly, with doubling times of around 2 weeks, and produce only very little biomass in batch cultures, successful proteogenomic, transcriptomic and biochemical studies revealed novel degradation pathways with exciting biochemical reactions such as for example the carboxylation of naphthalene or the ATP-independent reduction of naphthoyl-coenzyme A. The elucidation of the first anaerobic degradation pathways of naphthalene and methylnaphthalene at the genetic and biochemical level now opens the door to studying the anaerobic metabolism and ecology of anaerobic PAH degraders. This will contribute to assessing the fate of one of the most important contaminant classes in anoxic sediments and aquifers. © 2016 S. Karger AG, Basel.

  18. Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

    PubMed Central

    Huang, Ruiqi; O’Donnell, Andrew J.; Barboline, Jessica J.; Barkman, Todd J.

    2016-01-01

    Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study. PMID:27638206

  19. Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus Neurospora crassa Ergothioneine Biosynthetic Pathway

    PubMed Central

    2015-01-01

    Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible. PMID:25275953

  20. Thoughts on the Teaching of Metabolism

    ERIC Educational Resources Information Center

    Metzger, Robert P.

    2006-01-01

    Systems biology, metabolomics, metabolic engineering, and other recent developments in biochemistry suggest that future biochemists will require a detailed familiarity with the compounds and pathways of intermediary metabolism and their biochemical control. The challenge to the biochemistry instructor is the presentation of metabolic pathways in a…

  1. The Molecular Basis of Communication within the Cell.

    ERIC Educational Resources Information Center

    Berridge, Michael J.

    1985-01-01

    Only a few substances serve as signals within cells; this indicates that internal signal pathways are remarkably universal. The variety of physiological and biochemical processes regulated by known messengers is discussed along with chemical structures, pathways, inositol-lipid cycles, and cell growth regulation. (DH)

  2. Biochemical-Pathway Diversity in Archabacteria

    DTIC Science & Technology

    1988-06-28

    8a NAME OF_ FUNDINGISFF0N Gr ... FFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ORGANIZATION (If applicable) Office of Naval Researh ONR... BIOLOGY AND EVOLUTION OF MICROORGANISMS (July 24-28. 1989) in a talk entitled "Evolution of Metabolic Pathways". TRAINING ACTIVITIES: Dr. Raj Bhatnagar, a

  3. The fractional diffusion limit of a kinetic model with biochemical pathway

    NASA Astrophysics Data System (ADS)

    Perthame, Benoît; Sun, Weiran; Tang, Min

    2018-06-01

    Kinetic-transport equations that take into account the intracellular pathways are now considered as the correct description of bacterial chemotaxis by run and tumble. Recent mathematical studies have shown their interest and their relations to more standard models. Macroscopic equations of Keller-Segel type have been derived using parabolic scaling. Due to the randomness of receptor methylation or intracellular chemical reactions, noise occurs in the signaling pathways and affects the tumbling rate. Then comes the question to understand the role of an internal noise on the behavior of the full population. In this paper we consider a kinetic model for chemotaxis which includes biochemical pathway with noises. We show that under proper scaling and conditions on the tumbling frequency as well as the form of noise, fractional diffusion can arise in the macroscopic limits of the kinetic equation. This gives a new mathematical theory about how long jumps can be due to the internal noise of the bacteria.

  4. Design principles for enhancing phase sensitivity and suppressing phase fluctuations simultaneously in biochemical oscillatory systems.

    PubMed

    Fei, Chenyi; Cao, Yuansheng; Ouyang, Qi; Tu, Yuhai

    2018-04-12

    Biological systems need to function accurately in the presence of strong noise and at the same time respond sensitively to subtle external cues. Here we study design principles in biochemical oscillatory circuits to achieve these two seemingly incompatible goals. We show that energy dissipation can enhance phase sensitivity linearly by driving the phase-amplitude coupling and increase timing accuracy by suppressing phase diffusion. Two general design principles in the key underlying reaction loop formed by two antiparallel pathways are found to optimize oscillation performance with a given energy budget: balancing the forward-to-backward flux ratio between the two pathways to reduce phase diffusion and maximizing the net flux of the phase-advancing pathway relative to that of the phase-retreating pathway to enhance phase sensitivity. Experimental evidences consistent with these design principles are found in the circadian clock of cyanobacteria. Future experiments to test the predicted dependence of phase sensitivity on energy dissipation are proposed.

  5. Necrosome core machinery: MLKL.

    PubMed

    Zhang, Jing; Yang, Yu; He, Wenyan; Sun, Liming

    2016-06-01

    In the study of regulated cell death, the rapidly expanding field of regulated necrosis, in particular necroptosis, has been drawing much attention. The signaling of necroptosis represents a sophisticated form of a death pathway. Anti-caspase mechanisms (e.g., using inhibitors of caspases, or genetic ablation of caspase-8) switch cell fate from apoptosis to necroptosis. The initial extracellular death signals regulate RIP1 and RIP3 kinase activation. The RIP3-associated death complex assembly is necessary and sufficient to initiate necroptosis. MLKL was initially identified as an essential mediator of RIP1/RIP3 kinase-initiated necroptosis. Recent studies on the signal transduction using chemical tools and biomarkers support the idea that MLKL is able to make more functional sense for the core machinery of the necroptosis death complex, called the necrosome, to connect to the necroptosis execution. The experimental data available now have pointed that the activated MLKL forms membrane-disrupting pores causing membrane leakage, which extends the prototypical concept of morphological and biochemical events following necroptosis happening in vivo. The key role of MLKL in necroptosis signaling thus sheds light on the logic underlying this unique "membrane-explosive" cell death pathway. In this review, we provide the general concepts and strategies that underlie signal transduction of this form of cell death, and then focus specifically on the role of MLKL in necroptosis.

  6. Metabolomics, Standards, and Metabolic Modeling for Synthetic Biology in Plants

    PubMed Central

    Hill, Camilla Beate; Czauderna, Tobias; Klapperstück, Matthias; Roessner, Ute; Schreiber, Falk

    2015-01-01

    Life on earth depends on dynamic chemical transformations that enable cellular functions, including electron transfer reactions, as well as synthesis and degradation of biomolecules. Biochemical reactions are coordinated in metabolic pathways that interact in a complex way to allow adequate regulation. Biotechnology, food, biofuel, agricultural, and pharmaceutical industries are highly interested in metabolic engineering as an enabling technology of synthetic biology to exploit cells for the controlled production of metabolites of interest. These approaches have only recently been extended to plants due to their greater metabolic complexity (such as primary and secondary metabolism) and highly compartmentalized cellular structures and functions (including plant-specific organelles) compared with bacteria and other microorganisms. Technological advances in analytical instrumentation in combination with advances in data analysis and modeling have opened up new approaches to engineer plant metabolic pathways and allow the impact of modifications to be predicted more accurately. In this article, we review challenges in the integration and analysis of large-scale metabolic data, present an overview of current bioinformatics methods for the modeling and visualization of metabolic networks, and discuss approaches for interfacing bioinformatics approaches with metabolic models of cellular processes and flux distributions in order to predict phenotypes derived from specific genetic modifications or subjected to different environmental conditions. PMID:26557642

  7. Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

    PubMed Central

    Seigneurin-Berny, Daphné; Verdel, André; Curtet, Sandrine; Lemercier, Claudie; Garin, Jérôme; Rousseaux, Sophie; Khochbin, Saadi

    2001-01-01

    The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination. PMID:11689694

  8. A Continuum Model of Actin Waves in Dictyostelium discoideum

    PubMed Central

    Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.

    2013-01-01

    Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312

  9. Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

    PubMed

    Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

    2017-09-16

    Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly

    PubMed Central

    Cook, Jeremy D.; Kondapalli, Kalyan C.; Rawat, Swati; Childs, William C.; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L.

    2010-01-01

    Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich’s ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. PMID:20815377

  11. Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly

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

    Cook, J.; Kondapalli, K; Rawat, S

    2010-01-01

    Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less

  12. Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

    PubMed

    Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati; Childs, William C; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L

    2010-10-12

    Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

  13. The Stereochemistry of Biochemical Molecules: A Subject to Revisit

    ERIC Educational Resources Information Center

    Centelles, Josep J.; Imperial, Santiago

    2005-01-01

    Although Fischer's convention for stereoisomers is useful for simple molecules, the stereochemistry of complex biochemical molecules is often poorly indicated in textbooks. This article reports on errors in stereochemistry of complex hydrosoluble vitamin B12 molecule. Twenty-five popular biochemistry textbooks were examined for their treatment of…

  14. Activation of sorbitol pathway in metabolic syndrome and increased susceptibility to cataract in Wistar-Obese rats

    PubMed Central

    Giridharan, Nappan Veettil

    2012-01-01

    Purpose Obesity is a major public health problem worldwide, and of late, epidemiological studies indicate a preponderance of cataracts under obesity conditions. Although cataract is a multifactorial disorder and various biochemical mechanisms have been proposed, the influence of obesity on cataractogenesis has yet to be investigated. In such a scenario, a suitable animal model that develops cataract following the onset of obesity will be a welcome tool for biomedical research. Therefore, we investigated the molecular and biochemical basis for predisposition to cataract in the obese mutant rat models established in our institute because 15%–20% of these rats develop cataracts spontaneously as they reach 12–15 months of age. Methods We analyzed the major biochemical pathways in the normal lenses of different age groups of our obese mutant rat strains, Wistar/Obese (WNIN/Ob) and WNIN/GR-Ob, the former with euglycemia and the latter with an additional impaired glucose tolerance trait. In addition, sorbitol levels were estimated in the cataractous lenses of the obese rats. Results Except for the polyol pathway, all the principal pathways of the lens remained unaltered. Therefore, sorbitol levels were found to be high in the normal eye lenses of obese rats (WNIN/Ob and WNIN/GR-Ob) compared to their lean controls from three months of age onwards. Between WNIN/Ob and WNIN/GR-Ob, the levels of sorbitol were higher in the latter, suggesting a synergistic effect of impaired glucose tolerance along with obesity in the activation of the sorbitol pathway. Either way, an elevated sorbitol pathway seemed to be the predisposing factor responsible for cataract formation in these mutant rats. Conclusions Activation of the sorbitol pathway indeed enhances the risk of cataract development in conditions such as metabolic syndrome. These rat models thus may be valuable tools for investigating obesity-associated cataract and for developing intervention strategies, based on these findings. PMID:22393276

  15. Activation of sorbitol pathway in metabolic syndrome and increased susceptibility to cataract in Wistar-Obese rats.

    PubMed

    Reddy, Paduru Yadagiri; Giridharan, Nappan Veettil; Reddy, Geereddy Bhanuprakash

    2012-01-01

    Obesity is a major public health problem worldwide, and of late, epidemiological studies indicate a preponderance of cataracts under obesity conditions. Although cataract is a multifactorial disorder and various biochemical mechanisms have been proposed, the influence of obesity on cataractogenesis has yet to be investigated. In such a scenario, a suitable animal model that develops cataract following the onset of obesity will be a welcome tool for biomedical research. Therefore, we investigated the molecular and biochemical basis for predisposition to cataract in the obese mutant rat models established in our institute because 15%-20% of these rats develop cataracts spontaneously as they reach 12-15 months of age. We analyzed the major biochemical pathways in the normal lenses of different age groups of our obese mutant rat strains, Wistar/Obese (WNIN/Ob) and WNIN/GR-Ob, the former with euglycemia and the latter with an additional impaired glucose tolerance trait. In addition, sorbitol levels were estimated in the cataractous lenses of the obese rats. Except for the polyol pathway, all the principal pathways of the lens remained unaltered. Therefore, sorbitol levels were found to be high in the normal eye lenses of obese rats (WNIN/Ob and WNIN/GR-Ob) compared to their lean controls from three months of age onwards. Between WNIN/Ob and WNIN/GR-Ob, the levels of sorbitol were higher in the latter, suggesting a synergistic effect of impaired glucose tolerance along with obesity in the activation of the sorbitol pathway. Either way, an elevated sorbitol pathway seemed to be the predisposing factor responsible for cataract formation in these mutant rats. Activation of the sorbitol pathway indeed enhances the risk of cataract development in conditions such as metabolic syndrome. These rat models thus may be valuable tools for investigating obesity-associated cataract and for developing intervention strategies, based on these findings.

  16. Surface energetics and protein-protein interactions: analysis and mechanistic implications

    PubMed Central

    Peri, Claudio; Morra, Giulia; Colombo, Giorgio

    2016-01-01

    Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

  17. Exome sequencing identifies NFS1 deficiency in a novel Fe-S cluster disease, infantile mitochondrial complex II/III deficiency.

    PubMed

    Farhan, Sali M K; Wang, Jian; Robinson, John F; Lahiry, Piya; Siu, Victoria M; Prasad, Chitra; Kronick, Jonathan B; Ramsay, David A; Rupar, C Anthony; Hegele, Robert A

    2014-01-01

    Iron-sulfur (Fe-S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe-S proteins, to assist in various key biochemical pathways. Mutations in Fe-S proteins often disrupt Fe-S cluster assembly leading to a spectrum of severe disorders such as Friedreich's ataxia or iron-sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe-S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

  18. Complete Biosynthesis of Anthocyanins Using E. coli Polycultures.

    PubMed

    Jones, J Andrew; Vernacchio, Victoria R; Collins, Shannon M; Shirke, Abhijit N; Xiu, Yu; Englaender, Jacob A; Cress, Brady F; McCutcheon, Catherine C; Linhardt, Robert J; Gross, Richard A; Koffas, Mattheos A G

    2017-06-06

    Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies. IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts. Copyright © 2017 Jones et al.

  19. Engineering microbial electrocatalysis for chemical and fuel production.

    PubMed

    Rosenbaum, Miriam A; Henrich, Alexander W

    2014-10-01

    In many biotechnological areas, metabolic engineering and synthetic biology have become core technologies for biocatalyst development. Microbial electrocatalysis for biochemical and fuel production is still in its infancy and reactions rates and the product spectrum are currently very low. Therefore, molecular engineering strategies will be crucial for the advancement and realization of many new bioproduction routes using electroactive microorganisms. The complex and unresolved biochemistry and physiology of extracellular electron transfer and the lack of molecular tools for these new non-model hosts for genetic engineering constitute the major challenges for this effort. This review is providing an insight into the current status, challenges and promising approaches of pathway engineering for microbial electrocatalysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. The inverse problem of brain energetics: ketone bodies as alternative substrates

    NASA Astrophysics Data System (ADS)

    Calvetti, D.; Occhipinti, R.; Somersalo, E.

    2008-07-01

    Little is known about brain energy metabolism under ketosis, although there is evidence that ketone bodies have a neuroprotective role in several neurological disorders. We investigate the inverse problem of estimating reaction fluxes and transport rates in the different cellular compartments of the brain, when the data amounts to a few measured arterial venous concentration differences. By using a recently developed methodology to perform Bayesian Flux Balance Analysis and a new five compartment model of the astrocyte-glutamatergic neuron cellular complex, we are able to identify the preferred biochemical pathways during shortage of glucose and in the presence of ketone bodies in the arterial blood. The analysis is performed in a minimally biased way, therefore revealing the potential of this methodology for hypothesis testing.

  1. [Signal reception and processing by the retina].

    PubMed

    Eysel, U

    2007-01-01

    Phototransduction occurs in the retina, which, as an outsourced part of the brain, fulfills important tasks in neuronal processing for image analysis relevant to perception. Interlinked biochemical cycles with immense amplification factors transform the electromagnetic waves of light into neuronal activity, and photochemical adaptation allows adjustment to light intensities of over more than 10 logarithmic units. Beginning with its dual system of photoreceptors with highly sensible rods and a color sensitive cone system, the retina, with between 50 and 100 main cell types, is characterized by complex neuronal circuits. The resulting center-surround antagonism of the receptive fields serves, amongst other things, to amplify intensity and color contrasts. Specialized ganglion cell types give rise to parallel signaling pathways into the higher visual centers of the brain.

  2. Imaging of Lipids and Metabolites Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

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

    Lanekoff, Ingela; Laskin, Julia

    In recent years, mass spectroscopy imaging (MSI) has emerged as a foundational technique in metabolomics and drug screening providing deeper understanding of complex mechanistic pathways within biochemical systems and biological organisms. We have been invited to contribute a chapter to a new Springer series volume, entitled “Mass Spectrometry Imaging of Small Molecules”. The volume is planned for the highly successful lab protocol series Methods in Molecular Biology, published by Humana Press, USA. The volume is aimed to equip readers with step-by-step mass spectrometric imaging protocols and bring rapidly maturing methods of MS imaging to life science researchers. The chapter willmore » provide a detailed protocol of ambient MSI by use of nanospray desorption electrospray ionization.« less

  3. Applications of Two-Dimensional Electrophoresis Technology to the Study of Atherosclerosis

    PubMed Central

    Lepedda, Antonio J.

    2008-01-01

    Atherosclerosis is a multifactorial disease in which hypertension, diabetes, hyperlipidemia and other risk factors are thought to play a role. However, the molecular processes underlying plaque formation and progression are not yet completely known. In the last years some researchers applied proteomics technologies for the comprehension of biochemical pathways of atherogenesis and to search new cardiovascular biomarkers to be utilized either as early diagnostic traits or as targets for new drug therapies. Due to its intrinsic complexity, the problem has been approached by different strategies, all of which have some limitations. In this review, we summarize the most common critical experimental variables in two-dimensional electrophoresis-based techniques and recent data obtained by applying proteomic approaches in the study of atherosclerosis. PMID:27683313

  4. Engineering Escherichia coli coculture systems for the production of biochemical products.

    PubMed

    Zhang, Haoran; Pereira, Brian; Li, Zhengjun; Stephanopoulos, Gregory

    2015-07-07

    Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.

  5. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

    PubMed Central

    Shin, David S.; Pratt, Ashley J.; Tainer, John A.

    2014-01-01

    As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine. PMID:24701133

  6. Protoenzymes: the case of hyperbranched polyesters

    NASA Astrophysics Data System (ADS)

    Mamajanov, Irena; Cody, George D.

    2017-11-01

    Enzymes are biopolymeric complexes that catalyse biochemical reactions and shape metabolic pathways. Enzymes usually work with small molecule cofactors that actively participate in reaction mechanisms and complex, usually globular, polymeric structures capable of specific substrate binding, encapsulation and orientation. Moreover, the globular structures of enzymes possess cavities with modulated microenvironments, facilitating the progression of reaction(s). The globular structure is ensured by long folded protein or RNA strands. Synthesis of such elaborate complexes has proven difficult under prebiotically plausible conditions. We explore here that catalysis may have been performed by alternative polymeric structures, namely hyperbranched polymers. Hyperbranched polymers are relatively complex structures that can be synthesized under prebiotically plausible conditions; their globular structure is ensured by virtue of their architecture rather than folding. In this study, we probe the ability of tertiary amine-bearing hyperbranched polyesters to form hydrophobic pockets as a reaction-promoting medium for the Kemp elimination reaction. Our results show that polyesters formed upon reaction between glycerol, triethanolamine and organic acid containing hydrophobic groups, i.e. adipic and methylsuccinic acid, are capable of increasing the rate of Kemp elimination by a factor of up to 3 over monomeric triethanolamine. This article is part of the themed issue 'Reconceptualizing the origins of life'.

  7. Identification of an Mg2+-independent soluble phosphatidate phosphatase in cottonseed (Gossypium hirsutum L.)

    USDA-ARS?s Scientific Manuscript database

    Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the oil biosynthesis pathway in cottonseed. Towards understanding the biochemical pathway of oil accumulation in cottonseed, this study focused on phosphatidic acid phosphatase (PAP...

  8. Gluconeogenesis: An ancient biochemical pathway with a new twist

    PubMed Central

    Miyamoto, Tetsuya; Amrein, Hubert

    2017-01-01

    ABSTRACT Synthesis of sugars from simple carbon sources is critical for survival of animals under limited nutrient availability. Thus, sugar-synthesizing enzymes should be present across the entire metazoan spectrum. Here, we explore the evolution of glucose and trehalose synthesis using a phylogenetic analysis of enzymes specific for the two pathways. Our analysis reveals that the production of trehalose is the more ancestral biochemical process, found in single cell organisms and primitive metazoans, but also in insects. The gluconeogenic-specific enzyme glucose-6-phosphatase (G6Pase) first appears in Cnidaria, but is also present in Echinodermata, Mollusca and Vertebrata. Intriguingly, some species of nematodes and arthropods possess the genes for both pathways. Moreover, expression data from Drosophila suggests that G6Pase and, hence, gluconeogenesis, initially had a neuronal function. We speculate that in insects—and possibly in some vertebrates—gluconeogenesis may be used as a means of neuronal signaling. PMID:28121487

  9. Gluconeogenesis: An ancient biochemical pathway with a new twist.

    PubMed

    Miyamoto, Tetsuya; Amrein, Hubert

    2017-07-03

    Synthesis of sugars from simple carbon sources is critical for survival of animals under limited nutrient availability. Thus, sugar-synthesizing enzymes should be present across the entire metazoan spectrum. Here, we explore the evolution of glucose and trehalose synthesis using a phylogenetic analysis of enzymes specific for the two pathways. Our analysis reveals that the production of trehalose is the more ancestral biochemical process, found in single cell organisms and primitive metazoans, but also in insects. The gluconeogenic-specific enzyme glucose-6-phosphatase (G6Pase) first appears in Cnidaria, but is also present in Echinodermata, Mollusca and Vertebrata. Intriguingly, some species of nematodes and arthropods possess the genes for both pathways. Moreover, expression data from Drosophila suggests that G6Pase and, hence, gluconeogenesis, initially had a neuronal function. We speculate that in insects-and possibly in some vertebrates-gluconeogenesis may be used as a means of neuronal signaling.

  10. Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape

    PubMed Central

    Mattiazzi, M.; Jambhekar, A.; Kaferle, P.; DeRisi, J. L.; Križaj, I.

    2010-01-01

    Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A2 (PLA2s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA2 in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA2 activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA2 activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0533-8) contains supplementary material, which is available to authorized users. PMID:20379744

  11. TGF-{beta} signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

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

    Luciakova, Katarina, E-mail: katarina.luciakova@savba.sk; Kollarovic, Gabriel; Kretova, Miroslava

    2011-08-05

    Highlights: {yields} TGF-{beta} induces the formation of unique nuclear NF1/Smad4 complexes that repress expression of the ANT-2 gene. {yields} Repression is mediated through an NF1-dependent repressor element in the promoter. {yields} The formation of NF1/Smad4 complexes and the repression of ANT2 are prevented by inhibitors of p38 kinase and TGF-{beta} RI. {yields} NF1/Smad complexes implicate novel role for NF1 and Smad proteins in the regulation of growth. -- Abstract: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G.more » Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-{beta}, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-{beta} is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-{beta} are prevented by inhibitors of TGF-{beta} RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-{beta} and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-{beta} signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.« less

  12. S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway

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

    Mohan, Sepuru K.; Rani, Sandhya G.; Kumar, Sriramoju M.

    2009-03-13

    Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstratedmore » that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544-22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224-22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217-22223; C. Bagala, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041-1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca{sup 2+}-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing pathway, we have studied the interactions of S100A13 with C2A by {sup 1}H-{sup 15}N HSQC titration and 3D-filtered NOESY experiments. We characterized the binary complex structure of S100A13-C2A by using a variety of multi-dimensional NMR experiments. This complex acts as a template for FGF-1 dimerization and multiprotein complex formation.« less

  13. Structure, function, and engineering of enzymes in isoflavonoid biosynthesis.

    PubMed

    Wang, Xiaoqiang

    2011-03-01

    Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.

  14. Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria.

    PubMed

    Sánchez-Pascuala, Alberto; Nikel, Pablo I; de Lorenzo, Víctor

    2018-01-01

    The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (i.e., GlucoBrick) that facilitates their grouping in the form of functional modules that can be combined at the user's will. This novel genetic tool allows for the à la carte implementation or boosting of EMP pathway activities into different Gram-negative bacteria. The potential of the GlucoBrick platform is further illustrated by engineering novel glycolytic activities in the most representative members of the Pseudomonas genus (Pseudomonas putida and Pseudomonas aeruginosa).

  15. Detection of differentially expressed genes in broiler pectoralis major muscle affected by White Striping - Wooden Breast myopathies.

    PubMed

    Zambonelli, Paolo; Zappaterra, Martina; Soglia, Francesca; Petracci, Massimiliano; Sirri, Federico; Cavani, Claudio; Davoli, Roberta

    2016-12-01

    White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broiler fillets present an impaired visual appearance that negatively affects consumers' acceptability. Previous studies have highlighted in affected fillets a severely damaged muscle, showing profound inflammation, fibrosis, and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation, and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further defining the transcription patterns associated with these myopathies. © 2016 Poultry Science Association Inc.

  16. Renin Angiotensin Aldosterone System (RAAS): its biology and drug targets for treating diabetic nephropathy.

    PubMed

    Zain, Maryam; Awan, Fazli Rabbi

    2014-09-01

    Diabetes mellitus is a multifactorial disorder of hyperglycemia caused by a combination of biochemical, molecular and genetic factors, which leads to the dysfunction of various organs including kidneys. Diabetic nephropathy (DN) is one of the microvascular complications of diabetes that results due to poor glycemic control. Several molecular and biochemical pathways have been implicated in the pathogenesis of DN. Of these, the Renin Angiotensin Aldosterone System (RAAS) is considered as a key pathway. RAAS involves various subsystems which contribute to the development of DN. Mutations in several genes of the RAAS pathway have been associated with the development of DN. These genes or their products present them as therapeutic targets for potent drugs to control or prevent DN, and development of new drugs for targeting the RAAS. Drugs in use for DN are mainly the Angiotensin Converting Enzyme (ACE) inhibitors, Angiotensin Receptors Blockers (ARB) and renin inhibitors which play important roles in reducing DN. Hence, the present review is focused on the pathophysiology and genetic factors for DN by exploring the RAAS pathway and emphasizing the benefits of blocking this pathway to control and prevent DN.

  17. An improved hybrid of particle swarm optimization and the gravitational search algorithm to produce a kinetic parameter estimation of aspartate biochemical pathways.

    PubMed

    Ismail, Ahmad Muhaimin; Mohamad, Mohd Saberi; Abdul Majid, Hairudin; Abas, Khairul Hamimah; Deris, Safaai; Zaki, Nazar; Mohd Hashim, Siti Zaiton; Ibrahim, Zuwairie; Remli, Muhammad Akmal

    2017-12-01

    Mathematical modelling is fundamental to understand the dynamic behavior and regulation of the biochemical metabolisms and pathways that are found in biological systems. Pathways are used to describe complex processes that involve many parameters. It is important to have an accurate and complete set of parameters that describe the characteristics of a given model. However, measuring these parameters is typically difficult and even impossible in some cases. Furthermore, the experimental data are often incomplete and also suffer from experimental noise. These shortcomings make it challenging to identify the best-fit parameters that can represent the actual biological processes involved in biological systems. Computational approaches are required to estimate these parameters. The estimation is converted into multimodal optimization problems that require a global optimization algorithm that can avoid local solutions. These local solutions can lead to a bad fit when calibrating with a model. Although the model itself can potentially match a set of experimental data, a high-performance estimation algorithm is required to improve the quality of the solutions. This paper describes an improved hybrid of particle swarm optimization and the gravitational search algorithm (IPSOGSA) to improve the efficiency of a global optimum (the best set of kinetic parameter values) search. The findings suggest that the proposed algorithm is capable of narrowing down the search space by exploiting the feasible solution areas. Hence, the proposed algorithm is able to achieve a near-optimal set of parameters at a fast convergence speed. The proposed algorithm was tested and evaluated based on two aspartate pathways that were obtained from the BioModels Database. The results show that the proposed algorithm outperformed other standard optimization algorithms in terms of accuracy and near-optimal kinetic parameter estimation. Nevertheless, the proposed algorithm is only expected to work well in small scale systems. In addition, the results of this study can be used to estimate kinetic parameter values in the stage of model selection for different experimental conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Inferring genetic interactions via a nonlinear model and an optimization algorithm.

    PubMed

    Chen, Chung-Ming; Lee, Chih; Chuang, Cheng-Long; Wang, Chia-Chang; Shieh, Grace S

    2010-02-26

    Biochemical pathways are gradually becoming recognized as central to complex human diseases and recently genetic/transcriptional interactions have been shown to be able to predict partial pathways. With the abundant information made available by microarray gene expression data (MGED), nonlinear modeling of these interactions is now feasible. Two of the latest advances in nonlinear modeling used sigmoid models to depict transcriptional interaction of a transcription factor (TF) for a target gene, but do not model cooperative or competitive interactions of several TFs for a target. An S-shape model and an optimization algorithm (GASA) were developed to infer genetic interactions/transcriptional regulation of several genes simultaneously using MGED. GASA consists of a genetic algorithm (GA) and a simulated annealing (SA) algorithm, which is enhanced by a steepest gradient descent algorithm to avoid being trapped in local minimum. Using simulated data with various degrees of noise, we studied how GASA with two model selection criteria and two search spaces performed. Furthermore, GASA was shown to outperform network component analysis, the time series network inference algorithm (TSNI), GA with regular GA (GAGA) and GA with regular SA. Two applications are demonstrated. First, GASA is applied to infer a subnetwork of human T-cell apoptosis. Several of the predicted interactions are supported by the literature. Second, GASA was applied to infer the transcriptional factors of 34 cell cycle regulated targets in S. cerevisiae, and GASA performed better than one of the latest advances in nonlinear modeling, GAGA and TSNI. Moreover, GASA is able to predict multiple transcription factors for certain targets, and these results coincide with experiments confirmed data in YEASTRACT. GASA is shown to infer both genetic interactions and transcriptional regulatory interactions well. In particular, GASA seems able to characterize the nonlinear mechanism of transcriptional regulatory interactions (TIs) in yeast, and may be applied to infer TIs in other organisms. The predicted genetic interactions of a subnetwork of human T-cell apoptosis coincide with existing partial pathways, suggesting the potential of GASA on inferring biochemical pathways.

  19. Investigation on biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC-MS based metabolomics.

    PubMed

    Xu, Jie; Hu, Feng-Lin; Wang, Wei; Wan, Xiao-Chun; Bao, Guan-Hu

    2015-11-01

    Fu brick tea (FBT) is a unique post-fermented tea product which is fermented with fungi during the manufacturing process. In this study, we investigated the biochemical compositional changes occurring during the microbial fermentation process (MFP) of FBT based on non-targeted LC-MS, which was a comprehensive and unbiased methodology. Our data analysis took a two-phase approach: (1) comparison of FBT with other tea products using PCA analysis to exhibit the characteristic effect of MFP on the formation of Fu brick tea and (2) comparison of tea samples throughout the MFP of FBT to elucidate the possible key metabolic pathways produced by the fungi. Non-targeted LC-MS analysis clearly distinguished FBT with other tea samples and highlighted some interesting metabolic pathways during the MFP including B ring fission catechin. Our study demonstrated that those fungi had a significant influence on the biochemical profiles in the FBT and consequently contributed to its unique quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

    PubMed Central

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P.

    2016-01-01

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

  1. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes.

    PubMed

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P

    2016-08-30

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes.

  2. Biochemical Reconstitution of the WAVE Regulatory Complex

    PubMed Central

    Chen, Baoyu; Padrick, Shae B.; Henry, Lisa; Rosen, Michael K.

    2014-01-01

    The WAVE regulatory complex (WRC) is a 400-KDa heteropentameric protein assembly that plays a central role in controlling actin cytoskeletal dynamics in many cellular processes. The WRC acts by integrating diverse cellular cues and stimulating the actin nucleating activity of the Arp2/3 complex at membranes. Biochemical and biophysical studies of the underlying mechanisms of these processes require large amounts of purified WRC. Recent success in recombinant expression, reconstitution, purification and crystallization of the WRC has greatly advanced our understanding of the inhibition, activation and membrane recruitment mechanisms of this complex. But many important questions remain to be answered. Here we summarize and update the methods developed in our laboratory, which allow reliable and flexible production of tens of milligrams of recombinant WRC of crystallographic quality, sufficient for many biochemical and structural studies. PMID:24630101

  3. Biochemical and genetic analysis of Leigh syndrome patients in Korea.

    PubMed

    Chae, Jong-Hee; Lee, Jin Sook; Kim, Ki Joong; Hwang, Yong Seung; Hirano, Michio

    2008-06-01

    Sixteen Korean patients with Leigh syndrome were identified at the Seoul National University Children's Hospital in 2001-2006. Biochemical or molecular defects were identified in 14 patients (87.5%). Thirteen patients had respiratory chain enzyme defects; 9 had complex I deficiency, and 4 had combined defects of complex I+III+IV. Based on the biochemical defects, targeted genetic studies in 4 patients with complex I deficiency revealed two heteroplasmic mitochondrial DNA mutations in ND genes. One patient had the mitochondrial DNA T8993G point mutation. No mitochondrial DNA defects were identified in 11 (68.7%) of our LS patients, who probably have mutations in nuclear DNA. Although a limited study based in a single tertiary medical center, our findings suggest that isolated complex I deficiency may be the most common cause of Leigh syndrome in Korea.

  4. Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis[C][W

    PubMed Central

    Cobessi, David; Dumas, Renaud; Pautre, Virginie; Meinguet, Céline; Ferrer, Jean-Luc; Alban, Claude

    2012-01-01

    Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling. PMID:22547782

  5. DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

    PubMed

    Hovey, Raymond; Lentes, Sabine; Ehrenreich, Armin; Salmon, Kirsty; Saba, Karla; Gottschalk, Gerhard; Gunsalus, Robert P; Deppenmeier, Uwe

    2005-05-01

    Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.

  6. Elastic Network Model of a Nuclear Transport Complex

    NASA Astrophysics Data System (ADS)

    Ryan, Patrick; Liu, Wing K.; Lee, Dockjin; Seo, Sangjae; Kim, Young-Jin; Kim, Moon K.

    2010-05-01

    The structure of Kap95p was obtained from the Protein Data Bank (www.pdb.org) and analyzed RanGTP plays an important role in both nuclear protein import and export cycles. In the nucleus, RanGTP releases macromolecular cargoes from importins and conversely facilitates cargo binding to exportins. Although the crystal structure of the nuclear import complex formed by importin Kap95p and RanGTP was recently identified, its molecular mechanism still remains unclear. To understand the relationship between structure and function of a nuclear transport complex, a structure-based mechanical model of Kap95p:RanGTP complex is introduced. In this model, a protein structure is simply modeled as an elastic network in which a set of coarse-grained point masses are connected by linear springs representing biochemical interactions at atomic level. Harmonic normal mode analysis (NMA) and anharmonic elastic network interpolation (ENI) are performed to predict the modes of vibrations and a feasible pathway between locked and unlocked conformations of Kap95p, respectively. Simulation results imply that the binding of RanGTP to Kap95p induces the release of the cargo in the nucleus as well as prevents any new cargo from attaching to the Kap95p:RanGTP complex.

  7. PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer

    PubMed Central

    Caromile, Leslie Ann; Dortche, Kristina; Rahman, M. Mamunur; Grant, Christina L.; Stoddard, Christopher; Ferrer, Fernando A.; Shapiro, Linda H.

    2017-01-01

    Increased abundance of the prostate-specific membrane antigen (PSMA) on prostate epithelium is a hallmark of advanced metastatic prostate cancer (PCa) and correlates negatively with prognosis. However, direct evidence that PSMA functionally contributes to PCa progression remains elusive. We generated mice bearing PSMA-positive or PSMA-negative PCa by crossing PSMA-deficient mice with transgenic PCa (TRAMP) models, enabling direct assessment of PCa incidence and progression in the presence or absence of PSMA. Compared with PSMA-positive tumors, PSMA-negative tumors were smaller, lower-grade, and more apoptotic with fewer blood vessels, consistent with the recognized proangiogenic function of PSMA. Relative to PSMA-positive tumors, tumors lacking PSMA had less than half the abundance of type 1 insulin-like growth factor receptor (IGF-1R), less activity in the survival pathway mediated by PI3K-AKT signaling, and more activity in the proliferative pathway mediated by MAPK-ERK1/2 signaling. Biochemically, PSMA interacted with the scaffolding protein RACK1, disrupting signaling between the β1 integrin and IGF-1R complex to the MAPK pathway, enabling activation of the AKT pathway instead. Manipulation of PSMA abundance in PCa cell lines recapitulated this signaling pathway switch. Analysis of published databases indicated that IGF-1R abundance, cell proliferation, and expression of transcripts for antiapoptotic markers positively correlated with PSMA abundance in patients, suggesting that this switch may be relevant to human PCa. Our findings suggest that increase in PSMA in prostate tumors contributes to progression by altering normal signal transduction pathways to drive PCa progression and that enhanced signaling through the IGF-1R/β1 integrin axis may occur in other tumors. PMID:28292957

  8. In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

    PubMed Central

    Curatti, Leonardo; Hernandez, Jose A.; Igarashi, Robert Y.; Soboh, Basem; Zhao, Dehua; Rubio, Luis M.

    2007-01-01

    Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron–molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe2+, S2−, MoO42−, and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe2+, S2−, MoO42−, R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed. PMID:17978192

  9. Inhibition of the NEMO/IKKβ association complex formation, a novel mechanism associated with the NF-κB activation suppression by Withania somnifera's key metabolite withaferin A.

    PubMed

    Grover, Abhinav; Shandilya, Ashutosh; Punetha, Ankita; Bisaria, Virendra S; Sundar, Durai

    2010-12-02

    Nuclear Factor kappa B (NF-κB) is a transcription factor involved in the regulation of cell signaling responses and is a key regulator of cellular processes involved in the immune response, differentiation, cell proliferation, and apoptosis. The constitutive activation of NF-κB contributes to multiple cellular outcomes and pathophysiological conditions such as rheumatoid arthritis, asthma, inflammatory bowel disease, AIDS and cancer. Thus there lies a huge therapeutic potential beneath inhibition of NF-κB signalling pathway for reducing these chronic ailments. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show plethora of pharmacological activities like anti- inflammatory, antitumor, antibacterial, antioxidant, anticonvulsive, and immunosuppressive. Though a few studies have been reported depicting the effect of WA (withaferin A) on suppression of NF-κB activation, the mechanism behind this is still eluding the researchers. The study conducted here is an attempt to explore NF-κB signalling pathway modulating capability of Withania somnifera's major constituent WA and to elucidate its possible mode of action using molecular docking and molecular dynamics simulations studies. Formation of active IKK (IκB kinase) complex comprising NEMO (NF-κB Essential Modulator) and IKKβ subunits is one of the essential steps for NF-κB signalling pathway, non-assembly of which can lead to prevention of the above mentioned vulnerable disorders. As observed from our semi-flexible docking analysis, WA forms strong intermolecular interactions with the NEMO chains thus building steric as well as thermodynamic barriers to the incoming IKKβ subunits, which in turn pave way to naive complex formation capability of NEMO with IKKβ. Docking of WA into active NEMO/IKKβ complex using flexible docking in which key residues of the complex were kept flexible also suggest the disruption of the active complex. Thus the molecular docking analysis of WA into NEMO and active NEMO/IKKβ complex conducted in this study provides significant evidence in support of the proposed mechanism of NF-κB activation suppression by inhibition or disruption of active NEMO/IKKβ complex formation being accounted by non-assembly of the catalytically active NEMO/IKKβ complex. Results from the molecular dynamics simulations in water show that the trajectories of the native protein and the protein complexed with WA are stable over a considerably long time period of 2.6 ns. NF-κB is one of the most attractive topics in current biological, biochemical, and pharmacological research, and in the recent years the number of studies focusing on its inhibition/regulation has increased manifolds. Small ligands (both natural and synthetic) are gaining particular attention in this context. Our computational analysis provided a rationalization of the ability of naturally occurring withaferin A to alter the NF-κB signalling pathway along with its proposed mode of inhibition of the pathway. The absence of active IKK multisubunit complex would prevent degradation of IκB proteins, as the IκB proteins would not get phosphorylated by IKK. This would ultimately lead to non-release of NF-κB and its further translocation to the nucleus thus arresting its nefarious acts. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent NF-κB modulating capability. Moreover the present MD simulations made clear the dynamic structural stability of NEMO/IKKβ in complex with the drug WA, together with the inhibitory mechanism.

  10. Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

    PubMed Central

    Bebel, Aleksandra; Karaca, Ezgi; Kumar, Banushree; Stark, W Marshall; Barabas, Orsolya

    2016-01-01

    Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division. DOI: http://dx.doi.org/10.7554/eLife.19706.001 PMID:28009253

  11. Transition-Metal Hydride Radical Cations.

    PubMed

    Hu, Yue; Shaw, Anthony P; Estes, Deven P; Norton, Jack R

    2016-08-10

    Transition-metal hydride radical cations (TMHRCs) are involved in a variety of chemical and biochemical reactions, making a more thorough understanding of their properties essential for explaining observed reactivity and for the eventual development of new applications. Generally, these species may be treated as the ones formed by one-electron oxidation of diamagnetic analogues that are neutral or cationic. Despite the importance of TMHRCs, the generally sensitive nature of these complexes has hindered their development. However, over the last four decades, many more TMHRCs have been synthesized, characterized, isolated, or hypothesized as reaction intermediates. This comprehensive review focuses on experimental studies of TMHRCs reported through the year 2014, with an emphasis on isolated and observed species. The methods used for the generation or synthesis of TMHRCs are surveyed, followed by a discussion about the stability of these complexes. The fundamental properties of TMHRCs, especially those pertaining to the M-H bond, are described, followed by a detailed treatment of decomposition pathways. Finally, reactions involving TMHRCs as intermediates are described.

  12. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    PubMed

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

    2016-10-04

    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

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

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligandmore » positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.« less

  14. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    DOE PAGES

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; ...

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligandmore » positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.« less

  15. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    PubMed Central

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-01-01

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase, RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive due to uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules. PMID:25894083

  16. Stochasticity and bifurcations in a reduced model with interlinked positive and negative feedback loops of CREB1 and CREB2 stimulated by 5-HT.

    PubMed

    Hao, Lijie; Yang, Zhuoqin; Bi, Yuanhong

    2016-04-01

    The cyclic AMP (cAMP)-response element-binding protein (CREB) family of transcription factors is crucial in regulating gene expression required for long-term memory (LTM) formation. Upon exposure of sensory neurons to the neurotransmitter serotonin (5-HT), CREB1 is activated via activation of the protein kinase A (PKA) intracellular signaling pathways, and CREB2 as a transcriptional repressor is relieved possibly via phosphorylation of CREB2 by mitogen-activated protein kinase (MAPK). Song et al. [18] proposed a minimal model with only interlinked positive and negative feedback loops of transcriptional regulation by the activator CREB1 and the repressor CREB2. Without considering feedbacks between the CREB proteins, Pettigrew et al. [8] developed a computational model characterizing complex dynamics of biochemical pathways downstream of 5-HT receptors. In this work, to describe more simply the biochemical pathways and gene regulation underlying 5-HT-induced LTM, we add the important extracellular sensitizing stimulus 5-HT as well as the product Ap-uch into the Song's minimal model. We also strive to examine dynamical properties of the gene regulatory network under the changing concentration of the stimulus, [5-HT], cooperating with the varying positive feedback strength in inducing a high state of CREB1 for the establishment of long-term memory. Different dynamics including monostability, bistability and multistability due to coexistence of stable steady states and oscillations is investigated by means of codimension-2 bifurcation analysis. At the different positive feedback strengths, comparative analysis of deterministic and stochastic dynamics reveals that codimension-1 bifurcation with respect to [5-HT] as the parameter can predict diverse stochastic behaviors resulted from the finite number of molecules, and the number of CREB1 molecules more and more preferentially resides near the high steady state with increasing [5-HT], which contributes to long-term memory formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

    PubMed

    Casás-Selves, Matias; Zhang, Andrew X; Dowling, James E; Hallén, Stefan; Kawatkar, Aarti; Pace, Nicholas J; Denz, Christopher R; Pontz, Timothy; Garahdaghi, Farzin; Cao, Qing; Sabirsh, Alan; Thakur, Kumar; O'Connell, Nichole; Hu, Jun; Cornella-Taracido, Iván; Weerapana, Eranthie; Zinda, Michael; Goodnow, Robert A; Castaldi, M Paola

    2017-06-21

    Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca 2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

    PubMed

    Ledbetter, Rhesa N; Garcia Costas, Amaya M; Lubner, Carolyn E; Mulder, David W; Tokmina-Lukaszewska, Monika; Artz, Jacob H; Patterson, Angela; Magnuson, Timothy S; Jay, Zackary J; Duan, H Diessel; Miller, Jacquelyn; Plunkett, Mary H; Hoben, John P; Barney, Brett M; Carlson, Ross P; Miller, Anne-Frances; Bothner, Brian; King, Paul W; Peters, John W; Seefeldt, Lance C

    2017-08-15

    The biological reduction of dinitrogen (N 2 ) to ammonia (NH 3 ) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled to reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

  19. The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

    DOE PAGES

    Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E.; ...

    2017-07-13

    The biological reduction of dinitrogen (N 2) to ammonia (NH 3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled tomore » reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less

  20. RaMP: A Comprehensive Relational Database of Metabolomics Pathways for Pathway Enrichment Analysis of Genes and Metabolites

    PubMed Central

    Zhang, Bofei; Hu, Senyang; Baskin, Elizabeth; Patt, Andrew; Siddiqui, Jalal K.

    2018-01-01

    The value of metabolomics in translational research is undeniable, and metabolomics data are increasingly generated in large cohorts. The functional interpretation of disease-associated metabolites though is difficult, and the biological mechanisms that underlie cell type or disease-specific metabolomics profiles are oftentimes unknown. To help fully exploit metabolomics data and to aid in its interpretation, analysis of metabolomics data with other complementary omics data, including transcriptomics, is helpful. To facilitate such analyses at a pathway level, we have developed RaMP (Relational database of Metabolomics Pathways), which combines biological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, WikiPathways, and the Human Metabolome DataBase (HMDB). To the best of our knowledge, an off-the-shelf, public database that maps genes and metabolites to biochemical/disease pathways and can readily be integrated into other existing software is currently lacking. For consistent and comprehensive analysis, RaMP enables batch and complex queries (e.g., list all metabolites involved in glycolysis and lung cancer), can readily be integrated into pathway analysis tools, and supports pathway overrepresentation analysis given a list of genes and/or metabolites of interest. For usability, we have developed a RaMP R package (https://github.com/Mathelab/RaMP-DB), including a user-friendly RShiny web application, that supports basic simple and batch queries, pathway overrepresentation analysis given a list of genes or metabolites of interest, and network visualization of gene-metabolite relationships. The package also includes the raw database file (mysql dump), thereby providing a stand-alone downloadable framework for public use and integration with other tools. In addition, the Python code needed to recreate the database on another system is also publicly available (https://github.com/Mathelab/RaMP-BackEnd). Updates for databases in RaMP will be checked multiple times a year and RaMP will be updated accordingly. PMID:29470400

  1. RaMP: A Comprehensive Relational Database of Metabolomics Pathways for Pathway Enrichment Analysis of Genes and Metabolites.

    PubMed

    Zhang, Bofei; Hu, Senyang; Baskin, Elizabeth; Patt, Andrew; Siddiqui, Jalal K; Mathé, Ewy A

    2018-02-22

    The value of metabolomics in translational research is undeniable, and metabolomics data are increasingly generated in large cohorts. The functional interpretation of disease-associated metabolites though is difficult, and the biological mechanisms that underlie cell type or disease-specific metabolomics profiles are oftentimes unknown. To help fully exploit metabolomics data and to aid in its interpretation, analysis of metabolomics data with other complementary omics data, including transcriptomics, is helpful. To facilitate such analyses at a pathway level, we have developed RaMP (Relational database of Metabolomics Pathways), which combines biological pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, WikiPathways, and the Human Metabolome DataBase (HMDB). To the best of our knowledge, an off-the-shelf, public database that maps genes and metabolites to biochemical/disease pathways and can readily be integrated into other existing software is currently lacking. For consistent and comprehensive analysis, RaMP enables batch and complex queries (e.g., list all metabolites involved in glycolysis and lung cancer), can readily be integrated into pathway analysis tools, and supports pathway overrepresentation analysis given a list of genes and/or metabolites of interest. For usability, we have developed a RaMP R package (https://github.com/Mathelab/RaMP-DB), including a user-friendly RShiny web application, that supports basic simple and batch queries, pathway overrepresentation analysis given a list of genes or metabolites of interest, and network visualization of gene-metabolite relationships. The package also includes the raw database file (mysql dump), thereby providing a stand-alone downloadable framework for public use and integration with other tools. In addition, the Python code needed to recreate the database on another system is also publicly available (https://github.com/Mathelab/RaMP-BackEnd). Updates for databases in RaMP will be checked multiple times a year and RaMP will be updated accordingly.

  2. Biological monitoring of carcinogens: current status and perspectives.

    PubMed

    Pavanello, Sofia; Lotti, Marcello

    2012-04-01

    Biomonitoring exposures to carcinogens is common practice and a variety of biomarkers have been developed to assess both exposures and biochemical/biological effects. However, their clinical and preventive relevance is still uncertain. The understanding of cancer as a genetic disease has dramatically evolved during last decades, showing that cancer cell types acquire their characteristics with different strategies, time frames and microenvironments. Therefore, the place of current biomarkers within this complex scenario of gene-environment interactions leading to cancer cannot be defined. Reasons are manifold. Most studies assessed cancer risk on a group basis through snapshots taken at unknown time-points of the postulated chain of events. Little attention has been paid to the variety and variability of exposures, and no prospective study validated the indicators of biochemical/biological effects. New opportunities and suggestions for biomonitoring exposures to carcinogens could derive from exploring the exposome that combines exposures from all sources both external and internal. The discovery of new biomarkers and the identification of relevant gene-specific pathways could be achieved through metabolomic and genome-wide studies. In conclusion, it is possible to envisage personalized biomonitoring procedures, such as those already implemented in the context of nutrition and clinical oncology.

  3. Synthetic biology, inspired by synthetic chemistry.

    PubMed

    Malinova, V; Nallani, M; Meier, W P; Sinner, E K

    2012-07-16

    The topic synthetic biology appears still as an 'empty basket to be filled'. However, there is already plenty of claims and visions, as well as convincing research strategies about the theme of synthetic biology. First of all, synthetic biology seems to be about the engineering of biology - about bottom-up and top-down approaches, compromising complexity versus stability of artificial architectures, relevant in biology. Synthetic biology accounts for heterogeneous approaches towards minimal and even artificial life, the engineering of biochemical pathways on the organismic level, the modelling of molecular processes and finally, the combination of synthetic with nature-derived materials and architectural concepts, such as a cellular membrane. Still, synthetic biology is a discipline, which embraces interdisciplinary attempts in order to have a profound, scientific base to enable the re-design of nature and to compose architectures and processes with man-made matter. We like to give an overview about the developments in the field of synthetic biology, regarding polymer-based analogs of cellular membranes and what questions can be answered by applying synthetic polymer science towards the smallest unit in life, namely a cell. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Computational methods for diffusion-influenced biochemical reactions.

    PubMed

    Dobrzynski, Maciej; Rodríguez, Jordi Vidal; Kaandorp, Jaap A; Blom, Joke G

    2007-08-01

    We compare stochastic computational methods accounting for space and discrete nature of reactants in biochemical systems. Implementations based on Brownian dynamics (BD) and the reaction-diffusion master equation are applied to a simplified gene expression model and to a signal transduction pathway in Escherichia coli. In the regime where the number of molecules is small and reactions are diffusion-limited predicted fluctuations in the product number vary between the methods, while the average is the same. Computational approaches at the level of the reaction-diffusion master equation compute the same fluctuations as the reference result obtained from the particle-based method if the size of the sub-volumes is comparable to the diameter of reactants. Using numerical simulations of reversible binding of a pair of molecules we argue that the disagreement in predicted fluctuations is due to different modeling of inter-arrival times between reaction events. Simulations for a more complex biological study show that the different approaches lead to different results due to modeling issues. Finally, we present the physical assumptions behind the mesoscopic models for the reaction-diffusion systems. Input files for the simulations and the source code of GMP can be found under the following address: http://www.cwi.nl/projects/sic/bioinformatics2007/

  5. Biodiversity of genes encoding anti-microbial traits within plant associated microbes

    PubMed Central

    Mousa, Walaa K.; Raizada, Manish N.

    2015-01-01

    The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produces a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins, and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant-associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. We review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous coding sequences (CDS). We conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters. PMID:25914708

  6. Down-regulation of HSP60 Suppresses the Proliferation of Glioblastoma Cells via the ROS/AMPK/mTOR Pathway

    PubMed Central

    Tang, Haiping; Li, Jin; Liu, Xiaohui; Wang, Guihuai; Luo, Minkui; Deng, Haiteng

    2016-01-01

    Glioblastoma is a fatal and incurable cancer with the hyper-activated mTOR pathway. HSP60, a major chaperone for maintenance of mitochondrial proteostasis, is highly expressed in glioblastoma patients. To understand the effects of HSP60 on glioblastoma tumorigenesis and progression, we characterized the HSP60-knockdowned glioblastoma cells and revealed that HSP60 silencing markedly suppressed cell proliferation and promoted cell to undergo the epithelial-mesenchymal transition (EMT). Proteomic analysis showed that ribosomal proteins were significantly downregulated whereas EMT-associated proteins were up-regulated in HSP60-knockdowned U87 cells as confirmed by a distinct enrichment pattern in newly synthesized proteins with azido-homoalanine labeling. Biochemical analysis revealed that HSP60 knockdown increased reactive oxygen species (ROS) production that led to AMPK activation, similarly to the complex I inhibitor rotenone-induced AMPK activation. Activated AMPK suppressed mTORC1 mediated S6K and 4EBP1 phosphorylation to decrease protein translation, which slowed down cell growth and proliferation. On the other hand, high levels of ROS in HSP60 knockdowned or rotenone-treated U87 cells contributed to EMT. These results indicate that HSP60 silencing deactivates the mTOR pathway to suppress glioblastoma progression, suggesting that HSP60 is a potential therapeutic target for glioblastoma treatment. PMID:27325206

  7. Sphingolipid Signaling and Hematopoietic Malignancies: To the Rheostat and Beyond

    PubMed Central

    Loh, Kenneth C.; Baldwin, Dianna; Saba, Julie D.

    2011-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid with diverse functions including the promotion of cell survival, proliferation, and migration, as well as the regulation of angiogenesis, inflammation, immunity, vascular permeability and nuclear mechanisms that control gene transcription. S1P is derived from metabolism of ceramide, which itself has diverse and generally growth-inhibitory effects through its impact on downstream targets involved in regulation of apoptosis, senescence and cell cycle progression. Regulation of ceramide, S1P and the biochemical steps that modulate the balance and interconversion of these two lipids are major determinants of cell fate, a concept referred to as the “sphingolipid rheostat.” There is abundant evidence that the sphingolipid rheostat plays a role in the origination, progression and drug resistance patterns of hematopoietic malignancies. The pathway has also been exploited to circumvent the problem of chemotherapy resistance in leukemia and lymphoma. Given the broad effects of sphingolipids, targeting multiple steps in the metabolic pathway may provide possible therapeutic avenues. However, new observations have revealed that sphingolipid signaling effects are more complex than previously recognized, requiring a revision of the sphingolipid rheostat model. Here, we summarize recent insights regarding the sphingolipid metabolic pathway and its role in hematopoietic malignancies. PMID:21707493

  8. Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter.

    PubMed

    Oliveros, Alfredo; Starski, Phillip; Lindberg, Daniel; Choi, Sun; Heppelmann, Carrie J; Dasari, Surendra; Choi, Doo-Sup

    2017-04-07

    The neural circuit of the dorsal hippocampus (dHip) and nucleus accumbens (NAc) contributes to cue-induced learning and addictive behaviors, as demonstrated by the escalation of ethanol-seeking behaviors observed following deletion of the adenosine equilibrative nucleoside transporter 1 (ENT1 -/- ) in mice. Here we perform quantitative LC-MS/MS neuroproteomics in the dHip and NAc of ENT1 -/- mice. Using Ingenuity Pathway Analysis, we identified proteins associated with increased long-term potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein expression patterns suggesting deficits in glutamate degradation, GABAergic signaling, as well as significant changes in bioenergetics and energy homeostasis (oxidative phosphorylation, TCA cycle, and glycolysis). These pathways are consistent with previously reported behavioral and biochemical phenotypes that typify mice lacking ENT1. Moreover, we validated decreased expression of the SNARE complex protein VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin (dynorphin-A) in the NAc. Taken together, our proteomic approach provides novel pathways indicating that ENT1-regulated signaling is essential for neurotransmitter release and neuropeptide processing, both of which underlie learning and reward-seeking behaviors.

  9. The plant secretory pathway seen through the lens of the cell wall.

    PubMed

    van de Meene, A M L; Doblin, M S; Bacic, Antony

    2017-01-01

    Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.

  10. Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment

    PubMed Central

    Yang, Fu-Chun; Chen, Yi-Lung; Tang, Sen-Lin; Yu, Chang-Ping; Wang, Po-Hsiang; Ismail, Wael; Wang, Chia-Hsiang; Ding, Jiun-Yan; Yang, Cheng-Yu; Yang, Chia-Ying; Chiang, Yin-Ru

    2016-01-01

    Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative ‘omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available. PMID:26872041

  11. In Situ Tracking Kinetic Pathways of Li+/Na+ Substitution during Ion-Exchange Synthesis of LixNa1.5-xVOPO4F0.5.

    PubMed

    Park, Young-Uk; Bai, Jianming; Wang, Liping; Yoon, Gabin; Zhang, Wei; Kim, Hyungsub; Lee, Seongsu; Kim, Sung-Wook; Looney, J Patrick; Kang, Kisuk; Wang, Feng

    2017-09-13

    Ion exchange is a ubiquitous phenomenon central to wide industrial applications, ranging from traditional (bio)chemical separation to the emerging chimie douce synthesis of materials with metastable structure for batteries and other energy applications. The exchange process is complex, involving substitution and transport of different ions under non-equilibrium conditions, and thus difficult to probe, leaving a gap in mechanistic understanding of kinetic exchange pathways toward final products. Herein, we report in situ tracking kinetic pathways of Li + /Na + substitution during solvothermal ion-exchange synthesis of Li x Na 1.5-x VOPO 4 F 0.5 (0 ≤ x ≤ 1.5), a promising multi-Li polyanionic cathode for batteries. The real-time observation, corroborated by first-principles calculations, reveals a selective replacement of Na + by Li + , leading to peculiar Na + /Li + /vacancy orderings in the intermediates. Contradicting the traditional belief of facile topotactic substitution via solid solution reaction, an abrupt two-phase transformation occurs and predominantly governs the kinetics of ion exchange and transport in the 1D polyanionic framework, consequently leading to significant difference of Li stoichiometry and electrochemical properties in the exchanged products. The findings may help to pave the way for rational design of ion exchange synthesis for making new materials.

  12. Multiplatform serum metabolic phenotyping combined with pathway mapping to identify biochemical differences in smokers.

    PubMed

    Kaluarachchi, Manuja R; Boulangé, Claire L; Garcia-Perez, Isabel; Lindon, John C; Minet, Emmanuel F

    2016-10-01

    Determining perturbed biochemical functions associated with tobacco smoking should be helpful for establishing causal relationships between exposure and adverse events. A multiplatform comparison of serum of smokers (n = 55) and never-smokers (n = 57) using nuclear magnetic resonance spectroscopy, UPLC-MS and statistical modeling revealed clustering of the classes, distinguished by metabolic biomarkers. The identified metabolites were subjected to metabolic pathway enrichment, modeling adverse biological events using available databases. Perturbation of metabolites involved in chronic obstructive pulmonary disease, cardiovascular diseases and cancer were identified and discussed. Combining multiplatform metabolic phenotyping with knowledge-based mapping gives mechanistic insights into disease development, which can be applied to next-generation tobacco and nicotine products for comparative risk assessment.

  13. Lego bricks and the octet rule: Molecular models for biochemical pathways with plastic, interlocking toy bricks.

    PubMed

    Lin, Henry J; Lehoang, Jennifer; Kwan, Isabel; Baghaee, Anita; Prasad, Priya; Ha-Chen, Stephanie J; Moss, Tanesha; Woods, Jeremy D

    2018-01-01

    The 8 studs on a 2 × 4 Lego brick conveniently represent the outer shell of electrons for carbon, nitrogen, and oxygen atoms. We used Lego bricks to model these atoms, which are then joined together to form molecules by following the Lewis octet rule. A variety of small biological molecules can be modeled in this way, such as most amino acids, fatty acids, glucose, and various intermediate metabolites. Model building with these familiar toys can be a helpful, hands-on exercise for learning-or re-learning-biochemical pathways. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):54-57, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

  14. Oxidative DNA damage background estimated by a system model of base excision repair

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

    Sokhansanj, B A; Wilson, III, D M

    Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

  15. Assembly, organization, and function of the COPII coat

    PubMed Central

    Hughes, Helen

    2007-01-01

    A full mechanistic understanding of how secretory cargo proteins are exported from the endoplasmic reticulum for passage through the early secretory pathway is essential for us to comprehend how cells are organized, maintain compartment identity, as well as how they selectively secrete proteins and other macromolecules to the extracellular space. This process depends on the function of a multi-subunit complex, the COPII coat. Here we describe progress towards a full mechanistic understanding of COPII coat function, including the latest findings in this area. Much of our understanding of how COPII functions and is regulated comes from studies of yeast genetics, biochemical reconstitution and single cell microscopy. New developments arising from clinical cases and model organism biology and genetics enable us to gain far greater insight in to the role of membrane traffic in the context of a whole organism as well as during embryogenesis and development. A significant outcome of such a full understanding is to reveal how the machinery and processes of membrane trafficking through the early secretory pathway fail in disease states. PMID:18060556

  16. Heterosis: Many Genes, Many Mechanisms—End the Search for an Undiscovered Unifying Theory

    DOE PAGES

    Kaeppler, Shawn

    2012-01-01

    Heterosis is the increase in vigor that is observed in progenies of matings of diverse individuals from different species, isolated populations, or selected strains within species or populations. Heterosis has been of immense economic value in agriculture and has important implications regarding the fitness and fecundity of individuals in natural populations. Genetic models based on complementation of deleterious alleles, especially in the context of linkage and epistasis, are consistent with many observed manifestations of heterosis. The search for the genes and alleles that underlie heterosis, as well as for broader allele-independent, genomewide mechanisms, has encompassed many species and systems. Commonmore » themes across these studies indicate that sequence diversity is necessary but not sufficient to produce heterotic phenotypes, and that the molecular pathways that produce heterosis involve chromatin modification, transcriptional control, translation and protein processing, and interactions between and within developmental and biochemical pathways. Taken together, there are many and diverse molecular mechanisms that translate DNA into phenotype, and it is the combination of all these mechanisms across many genes that produce heterosis in complex traits.« less

  17. Epigenetics and Epigenomics of Plants.

    PubMed

    Yadav, Chandra Bhan; Pandey, Garima; Muthamilarasan, Mehanathan; Prasad, Manoj

    2018-01-23

    The genetic material DNA in association with histone proteins forms the complex structure called chromatin, which is prone to undergo modification through certain epigenetic mechanisms including cytosine DNA methylation, histone modifications, and small RNA-mediated methylation. Alterations in chromatin structure lead to inaccessibility of genomic DNA to various regulatory proteins such as transcription factors, which eventually modulates gene expression. Advancements in high-throughput sequencing technologies have provided the opportunity to study the epigenetic mechanisms at genome-wide levels. Epigenomic studies using high-throughput technologies will widen the understanding of mechanisms as well as functions of regulatory pathways in plant genomes, which will further help in manipulating these pathways using genetic and biochemical approaches. This technology could be a potential research tool for displaying the systematic associations of genetic and epigenetic variations, especially in terms of cytosine methylation onto the genomic region in a specific cell or tissue. A comprehensive study of plant populations to correlate genotype to epigenotype and to phenotype, and also the study of methyl quantitative trait loci (QTL) or epiGWAS, is possible by using high-throughput sequencing methods, which will further accelerate molecular breeding programs for crop improvement. Graphical Abstract.

  18. Inflammation in Alzheimer's Disease and Molecular Genetics: Recent Update.

    PubMed

    Zhang, Zhi-Gang; Li, Yan; Ng, Cheung Toa; Song, You-Qiang

    2015-10-01

    Alzheimer's disease (AD) is a complex age-related neurodegenerative disorder of the central nervous system. Since the first description of AD in 1907, many hypotheses have been established to explain its causes. The inflammation theory is one of them. Pathological and biochemical studies of brains from AD individuals have provided solid evidence of the activation of inflammatory pathways. Furthermore, people with long-term medication of anti-inflammatory drugs have shown a reduced risk to develop the disease. After three decades of genetic study in AD, dozens of loci harboring genetic variants influencing inflammatory pathways in AD patients has been identified through genome-wide association studies (GWAS). The most well-known GWAS risk factor that is responsible for immune response and inflammation in AD development should be APOE ε4 allele. However, a growing number of other GWAS risk AD candidate genes in inflammation have recently been discovered. In the present study, we try to review the inflammation in AD and immunity-associated GWAS risk genes like HLA-DRB5/DRB1, INPP5D, MEF2C, CR1, CLU and TREM2.

  19. Solution state nuclear magnetic resonance spectroscopy for biological metabolism and pathway intermediate analysis.

    PubMed

    Nealon, Gareth L; Howard, Mark J

    2016-12-15

    Using nuclear magnetic resonance (NMR) spectroscopy in the study of metabolism has been immensely popular in medical- and health-related research but has yet to be widely applied to more fundamental biological problems. This review provides some NMR background relevant to metabolism, describes why 1 H NMR spectra are complex as well as introducing relevant terminology and definitions. The applications and practical considerations of NMR metabolic profiling and 13 C NMR-based flux analyses are discussed together with the elegant 'enzyme trap' approach for identifying novel metabolic pathway intermediates. The importance of sample preparation and data analysis are also described and explained with reference to data precision and multivariate analysis to introduce researchers unfamiliar with NMR and metabolism to consider this technique for their research interests. Finally, a brief glance into the future suggests NMR-based metabolism has room to expand in the 21st century through new isotope labels, and NMR technologies and methodologies. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  20. PATHOPHYSIOLOGY AND THE CARDIORENAL CONNECTION IN HEART FAILURE. CIRCULATING HORMONES: BIOMARKERS OR MEDIATORS

    PubMed Central

    BUGLIONI, ALESSIA; BURNETT, JOHN C.

    2014-01-01

    Heart failure (HF) is a syndrome characterized by a complex pathophysiology which involves multiple organ systems, with the kidney playing a major role. HF can present with reduced ejection fraction (EF), HFrEF, or with preserved EF (HFpEF). The interplay between diverse organ systems contributing to HF is mediated by the activation of counteracting neurohormonal pathways focused to re-establishing hemodynamic homeostasis. During early stages of HF, these biochemical signals, consisting mostly of hormones and neurotransmitters secreted by a variety of cell types, are compensatory and the patient is asymptomatic. However, with disease progression, the attempt to reverse or delay cardiac dysfunction is deleterious, leading to multi-organ congestion, fibrosis and decompensation and finally symptomatic HF. In conclusion, these neurohormonal pathways mediate the evolution of HF and have become a way to monitor HF. Specifically, these mediators have become important in the diagnosis and prognosis of this highly fatal cardiovascular disease. Finally, while these multiple neurohumoral factors serve as important HF biomarkers, they can also be targeted for more effective and curative HF treatments. PMID:25445413

  1. Current Status on Biochemistry and Molecular Biology of Microbial Degradation of Nicotine

    PubMed Central

    Gurusamy, Raman; Natarajan, Sakthivel

    2013-01-01

    Bioremediation is one of the most promising methods to clean up polluted environments using highly efficient potent microbes. Microbes with specific enzymes and biochemical pathways are capable of degrading the tobacco alkaloids including highly toxic heterocyclic compound, nicotine. After the metabolic conversion, these nicotinophilic microbes use nicotine as the sole carbon, nitrogen, and energy source for their growth. Various nicotine degradation pathways such as demethylation pathway in fungi, pyridine pathway in Gram-positive bacteria, pyrrolidine pathway, and variant of pyridine and pyrrolidine pathways in Gram-negative bacteria have been reported. In this review, we discussed the nicotine-degrading pathways of microbes and their enzymes and biotechnological applications of nicotine intermediate metabolites. PMID:24470788

  2. SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

    PubMed

    Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

    2004-10-01

    The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

  3. Dark fermentation of complex waste biomass for biohydrogen production by pretreated thermophilic anaerobic digestate.

    PubMed

    Ghimire, Anish; Frunzo, Luigi; Pontoni, Ludovico; d'Antonio, Giuseppe; Lens, Piet N L; Esposito, Giovanni; Pirozzi, Francesco

    2015-04-01

    The Biohydrogen Potential (BHP) of six different types of waste biomass typical for the Campania Region (Italy) was investigated. Anaerobic sludge pre-treated with the specific methanogenic inhibitor sodium 2-bromoethanesulfonic acid (BESA) was used as seed inoculum. The BESA pre-treatment yielded the highest BHP in BHP tests carried out with pre-treated anaerobic sludge using potato and pumpkin waste as the substrates, in comparison with aeration or heat shock pre-treatment. The BHP tests carried out with different complex waste biomass showed average BHP values in a decreasing order from potato and pumpkin wastes (171.1 ± 7.3 ml H2/g VS) to buffalo manure (135.6 ± 4.1 ml H2/g VS), dried blood (slaughter house waste, 87.6 ± 4.1 ml H2/g VS), fennel waste (58.1 ± 29.8 ml H2/g VS), olive pomace (54.9 ± 5.4 ml H2/g VS) and olive mill wastewater (46.0 ± 15.6 ml H2/g VS). The digestate was analyzed for major soluble metabolites to elucidate the different biochemical pathways in the BHP tests. These showed the H2 was produced via mixed type fermentation pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi

    PubMed Central

    Rodríguez-Romero, J.; Franceschetti, M.; Bueno, E.; Sesma, A.

    2015-01-01

    Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs. PMID:25514925

  5. SIMULTANEOUS PRODUCTION OF TWO CAPSULAR POLYSACCHARIDES BY PNEUMOCOCCUS

    PubMed Central

    Austrian, Robert; Bernheimer, Harriet P.; Smith, Evelyn E. B.; Mills, George T.

    1959-01-01

    Study of the capsular genome of pneumococcus has shown that it controls a multiplicity of biochemical reactions essential to the synthesis of capsular polysaccharide. Mutation affecting any one of several biochemical reactions concerned with capsular synthesis may result in loss of capsulation without alteration of other biochemical functions similarly concerned. Mutations affecting the synthesis of uronic acids are an important cause of loss of capsulation and of virulence by strains of pneumococcus Type I and Type III. The capsular genome appears to have a specific location in the total genome of the cell, this locus being occupied by the capsular genome of whatever capsular type is expressed by the cell. Transformation of capsulated or of non-capsulated pneumococci to heterologous capsular type results probably from a genetic exchange followed by the development of a new biosynthetic pathway in the transformed cell. The new capsular genome is transferred to the transformed cell as a single particle of DNA. Binary capsulation results from the simultaneous presence within the pneumococcal cell of two capsular genomes, one mutated, the other normal. Interaction between the biochemical pathways controlled by the two capsular genomes leads to augmentation of the phenotypic expression of the product controlled by one and to partial suppression of the product determined by the other. Knowledge of the biochemical basis of binary capsulation can be used to indicate the presence of uronic acid in the capsular polysaccharide of a pneurnococcal type the composition of the capsule of which is unknown. PMID:13795197

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

    Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E.

    The biological reduction of dinitrogen (N 2) to ammonia (NH 3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E m = -320 mV) coupled tomore » reduction of flavodoxin semiquinone (E m = -460 mV) and reduction of coenzyme Q (E m = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less

  7. Engineering Escherichia coli coculture systems for the production of biochemical products

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

    Zhang, Haoran; Pereira, Brian; Li, Zhengjun

    Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. Furthermore, by efficiently producing another compound, 4-hydroxybenzoic acid, wemore » also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.« less

  8. Spatio-mechanical EphA2/ephrin-A1 Signaling in Cancer Cells

    NASA Astrophysics Data System (ADS)

    Xu, Qian

    2011-12-01

    Communication strategies in nature are an integral part to the survival of multi-cellular organisms. Cell membranes provide the chemical environment in which intercellular signaling begins. The vast complexity of this signaling requires that a relatively conserved set of chemical constituents be able to generate enormous signal diversity. Spatial sorting of signaling molecules within the membrane allows for this diversity. My research uses synthetic lipid membranes, solid-state nanostructures, and high-resolution imaging to study a potentially novel spatio-mechanical regulatory mechanism in the EphA2 signaling pathway. My hypothesis is that the multi-scale organization of the EphA2 receptor in the cell membrane regulates its biochemical function. This hypothesis is motivated by the idea that extracellular mechanical inputs have an important role in intracellular signaling cascades.

  9. Unveiling the functional diversity of the Alpha-Beta hydrolase fold in plants

    PubMed Central

    Mindrebo, Jeffrey T.; Nartey, Charisse M.; Seto, Yoshiya; Burkart, Michael D.; Noel, Joseph P.

    2017-01-01

    The alpha/beta hydrolase (ABH) superfamily is a widespread and functionally malleable protein fold recognized for its diverse biochemical activities across all three domains of life. ABH enzymes possess unexpected catalytic activity in the green plant lineage through selective alterations in active site architecture and chemistry. Furthermore, the ABH fold serves as the core structure for phytohormone and ligand receptors in the gibberellin, strigolactone, and karrikin signaling pathways in plants. Despite recent discoveries, the ABH family is sparsely characterized in plants, a sessile kingdom known to evolve complex and specialized chemical adaptations as survival responses to widely varying biotic and abiotic ecologies. This review calls attention to the ABH superfamily in the plant kingdom to highlight the functional adaptability of the ABH fold. PMID:27662376

  10. Hormonal control of cold stress responses in plants.

    PubMed

    Eremina, Marina; Rozhon, Wilfried; Poppenberger, Brigitte

    2016-02-01

    Cold stress responses in plants are highly sophisticated events that alter the biochemical composition of cells for protection from damage caused by low temperatures. In addition, cold stress has a profound impact on plant morphologies, causing growth repression and reduced yields. Complex signalling cascades are utilised to induce changes in cold-responsive gene expression that enable plants to withstand chilling or even freezing temperatures. These cascades are governed by the activity of plant hormones, and recent research has provided a better understanding of how cold stress responses are integrated with developmental pathways that modulate growth and initiate other events that increase cold tolerance. Information on the hormonal control of cold stress signalling is summarised to highlight the significant progress that has been made and indicate gaps that still exist in our understanding.

  11. DNA-Based Enzyme Reactors and Systems

    PubMed Central

    Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.

    2016-01-01

    During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. PMID:28335267

  12. Engineering Escherichia coli coculture systems for the production of biochemical products

    DOE PAGES

    Zhang, Haoran; Pereira, Brian; Li, Zhengjun; ...

    2015-06-25

    Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. Furthermore, by efficiently producing another compound, 4-hydroxybenzoic acid, wemore » also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.« less

  13. Mechanism and regulation of NLRP3 inflammasome activation

    PubMed Central

    He, Yuan; Hara, Hideki; Núñez, Gabriel

    2016-01-01

    Members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family and the pyrin and HIN-domain (PYHIN) family can form multiprotein complexes termed “inflammasomes”. The biochemical function of inflammasomes is to activate caspase-1, which leads to the maturation of interleukin 1β (IL-1β) and IL-18 and induction of pyroptosis, a form of cell death. Unlike other inflammasomes, the NLRP3 inflammasome can be activated by diverse stimuli. The importance of the NLRP3 inflammasome in immunity and human diseases has been well documented, but the mechanism and regulation of NLRP3 inflammasome activation remains unclear. In this review we summarize current understanding of the mechanism and regulation of NLRP3 inflammasome activation, as well as recent advances in the non-canonical and alternative inflammasome pathways. PMID:27669650

  14. The cellular mastermind(?) – Mechanotransduction and the nucleus

    PubMed Central

    Kaminski, Ashley; Fedorchak, Gregory R.; Lammerding, Jan

    2015-01-01

    Cells respond to mechanical stimulation by activation of specific signaling pathways and genes that allow the cell to adapt to its dynamic physical environment. How cells sense the various mechanical inputs and translate them into biochemical signals remains an area of active investigation. Recent reports suggest that the cell nucleus may be directly implicated in this cellular mechanotransduction process. In this chapter, we discuss how forces applied to the cell surface and cytoplasm induce changes in nuclear structure and organization, which could directly affect gene expression, while also highlighting the complex interplay between nuclear structural proteins and transcriptional regulators that may further modulate mechanotransduction signaling. Taken together, these findings paint a picture of the nucleus as a central hub in cellular mechanotransduction—both structurally and biochemically—with important implications in physiology and disease. PMID:25081618

  15. Protein function in precision medicine: deep understanding with machine learning.

    PubMed

    Rost, Burkhard; Radivojac, Predrag; Bromberg, Yana

    2016-08-01

    Precision medicine and personalized health efforts propose leveraging complex molecular, medical and family history, along with other types of personal data toward better life. We argue that this ambitious objective will require advanced and specialized machine learning solutions. Simply skimming some low-hanging results off the data wealth might have limited potential. Instead, we need to better understand all parts of the system to define medically relevant causes and effects: how do particular sequence variants affect particular proteins and pathways? How do these effects, in turn, cause the health or disease-related phenotype? Toward this end, deeper understanding will not simply diffuse from deeper machine learning, but from more explicit focus on understanding protein function, context-specific protein interaction networks, and impact of variation on both. © 2016 Federation of European Biochemical Societies.

  16. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.

    PubMed

    Chendrimada, Thimmaiah P; Gregory, Richard I; Kumaraswamy, Easwari; Norman, Jessica; Cooch, Neil; Nishikura, Kazuko; Shiekhattar, Ramin

    2005-08-04

    MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

  17. Evidence from Biochemical Pathways in Favor of Unfinished Evolution Rather than Intelligent Design

    ERIC Educational Resources Information Center

    Behrman, Edward J.; Marzluf, George A.

    2004-01-01

    An argument is made in favor of imperfect or unfinished evolution based on some metabolic pathways in which it seems that intelligent design would have done better. The case studies noted indicate the absence of highly intelligent design and are not intended as comprehensive collection but as a limited sample of inefficient situations in…

  18. genome-wide association and metabolic pathway analysis of corn earworm resistance in maize

    Treesearch

    Marilyn L. Warburton; Erika D. Womack; Juliet D. Tang; Adam Thrash; J. Spencer Smith; Wenwei Xu; Seth C. Murray; W. Paul Williams

    2018-01-01

    Maize (Zea mays mays L.) is a staple crop of economic, industrial, and food security importance. Damage to the growing ears by corn earworm [Helicoverpa zea (Boddie)] is a major economic burden and increases secondary fungal infections and mycotoxin levels. To identify biochemical pathways associated with native resistance mechanisms, a genome-wide...

  19. Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP).

    PubMed

    Wang, Le; Liu, Yi-Tong; Hao, Rui; Chen, Lei; Chang, Zhijie; Wang, Hong-Rui; Wang, Zhi-Xin; Wu, Jia-Wei

    2011-05-06

    The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

  20. Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3-MSP1 complex.

    PubMed

    Baldwin, Michael; Yamodo, Innocent; Ranjan, Ravi; Li, Xuerong; Mines, Gregory; Marinkovic, Marina; Hanada, Toshihiko; Oh, Steven S; Chishti, Athar H

    2014-12-01

    Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP119 and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Accurate atom-mapping computation for biochemical reactions.

    PubMed

    Latendresse, Mario; Malerich, Jeremiah P; Travers, Mike; Karp, Peter D

    2012-11-26

    The complete atom mapping of a chemical reaction is a bijection of the reactant atoms to the product atoms that specifies the terminus of each reactant atom. Atom mapping of biochemical reactions is useful for many applications of systems biology, in particular for metabolic engineering where synthesizing new biochemical pathways has to take into account for the number of carbon atoms from a source compound that are conserved in the synthesis of a target compound. Rapid, accurate computation of the atom mapping(s) of a biochemical reaction remains elusive despite significant work on this topic. In particular, past researchers did not validate the accuracy of mapping algorithms. We introduce a new method for computing atom mappings called the minimum weighted edit-distance (MWED) metric. The metric is based on bond propensity to react and computes biochemically valid atom mappings for a large percentage of biochemical reactions. MWED models can be formulated efficiently as Mixed-Integer Linear Programs (MILPs). We have demonstrated this approach on 7501 reactions of the MetaCyc database for which 87% of the models could be solved in less than 10 s. For 2.1% of the reactions, we found multiple optimal atom mappings. We show that the error rate is 0.9% (22 reactions) by comparing these atom mappings to 2446 atom mappings of the manually curated Kyoto Encyclopedia of Genes and Genomes (KEGG) RPAIR database. To our knowledge, our computational atom-mapping approach is the most accurate and among the fastest published to date. The atom-mapping data will be available in the MetaCyc database later in 2012; the atom-mapping software will be available within the Pathway Tools software later in 2012.

  2. Bio-Spectroscopic Imaging Provides Evidence of Hippocampal Zn Deficiency and Decreased Lipid Unsaturation in an Accelerated Ageing Mouse Model.

    PubMed

    Fimognari, Nicholas; Hollings, Ashley; Lam, Virginie; Tidy, Rebecca J; Kewish, Cameron M; Albrecht, Matthew A; Takechi, Ryu; Mamo, John C L; Hackett, Mark J

    2018-06-14

    Western society is facing a health epidemic due to the increasing incidence of dementia in ageing populations, and there are still few effective diagnostic methods, minimal treatment options, and no cure. Ageing is the greatest risk factor for memory loss that occurs during the natural ageing process, as well as being the greatest risk factor for neurodegenerative disease such as Alzheimer's disease. Therefore, greater understanding of the biochemical pathways that drive a healthy ageing brain towards dementia (pathological ageing or Alzheimer's disease), is required to accelerate the development of improved diagnostics and therapies. Unfortunately, many animal models of dementia model chronic amyloid precursor protein over-expression, which although highly relevant to mechanisms of amyloidosis and familial Alzheimer's disease, does not model well dementia during the natural ageing process. A promising animal model reported to model mechanisms of accelerated natural ageing and memory impairments, is the senescence accelerated murine prone strain 8 (SAMP8), which has been adopted by many research group to study the biochemical transitions that occur during brain ageing. A limitation to traditional methods of biochemical characterisation is that many important biochemical and elemental markers (lipid saturation, lactate, transition metals) cannot be imaged at meso- or micro-spatial resolution. Therefore, in this investigation we report the first multi-modal biospectroscopic characterisation of the SAMP8 model, and have identified important biochemical and elemental alterations, and co-localisations, between 4 month old SAMP8 mice and the relevant control (SAMR1) mice. Specifically, we demonstrate direct evidence of altered metabolism and disturbed lipid homeostasis within corpus callosum white matter, in addition to localised hippocampal metal deficiencies, in the accelerated ageing phenotype. Such findings have important implication for future research aimed at elucidating specific biochemical pathways for therapeutic intervention.

  3. Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV).

    PubMed

    Varela, Anna Lidia N; Komatsu, Setsuko; Wang, Xin; Silva, Rodolpho G G; Souza, Pedro Filho N; Lobo, Ana Karla M; Vasconcelos, Ilka M; Silveira, Joaquim A G; Oliveira, Jose T A

    2017-06-23

    Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Comparative clinical study between the effect of fenofibrate alone and its combination with pentoxifylline on biochemical parameters and liver stiffness in patients with non-alcoholic fatty liver disease.

    PubMed

    El-Haggar, Sahar Mohamed; Mostafa, Tarek Mohamed

    2015-07-01

    Non-alcoholic fatty liver disease is a common health problem associated with increased liver and vascular specific complications. The purpose of this study was to assess and compare the effect of fenofibrate alone or in combination with pentoxifylline on the measured biochemical parameters, inflammatory pathway and liver stiffness in patients with non-alcoholic fatty liver disease. The study design was randomized controlled trial. From July 2013 to June 2014, we recruited 90 non-alcoholic fatty liver patients from the Internal Medicine Department at Tanta University Hospital, Egypt. They were classified randomly into two groups to receive fenofibrate 300 mg daily or fenofibrate 300 mg daily plus pentoxifylline 1200 mg/day in three divided doses for 24 weeks. Fasting blood sample was obtained before and 24 weeks after treatment for biochemical analysis of liver and lipid panels, tumor necrosis factor-alpha, hyaluronic acid, transforming growth factor beta 1, fasting plasma insulin and fasting glucose. Liver stiffness measurement was carried out using fibro-scan. Data were statistically analyzed by paired and unpaired Student's t test. The data obtained suggests that adding pentoxifylline to fenofibrate does not provide a beneficial effect on lipid panel, but has a beneficial effect on indirect biochemical markers of hepatic fibrosis, a direct marker linked to matrix deposition (hyaluronic acid), a cytokine/growth factor linked to liver fibrosis (transforming growth factor beta 1), the inflammatory pathway, insulin resistance and liver stiffness as compared to fenofibrate alone. The combination pentoxifylline plus fenofibrate may represent a new therapeutic strategy for non-alcoholic fatty liver disease as it resulted in more beneficial effects on direct and indirect markers of liver fibrosis, liver stiffness, insulin resistance and inflammatory pathway implicated in NAFLD.

  5. Sig2BioPAX: Java tool for converting flat files to BioPAX Level 3 format.

    PubMed

    Webb, Ryan L; Ma'ayan, Avi

    2011-03-21

    The World Wide Web plays a critical role in enabling molecular, cell, systems and computational biologists to exchange, search, visualize, integrate, and analyze experimental data. Such efforts can be further enhanced through the development of semantic web concepts. The semantic web idea is to enable machines to understand data through the development of protocol free data exchange formats such as Resource Description Framework (RDF) and the Web Ontology Language (OWL). These standards provide formal descriptors of objects, object properties and their relationships within a specific knowledge domain. However, the overhead of converting datasets typically stored in data tables such as Excel, text or PDF into RDF or OWL formats is not trivial for non-specialists and as such produces a barrier to seamless data exchange between researchers, databases and analysis tools. This problem is particularly of importance in the field of network systems biology where biochemical interactions between genes and their protein products are abstracted to networks. For the purpose of converting biochemical interactions into the BioPAX format, which is the leading standard developed by the computational systems biology community, we developed an open-source command line tool that takes as input tabular data describing different types of molecular biochemical interactions. The tool converts such interactions into the BioPAX level 3 OWL format. We used the tool to convert several existing and new mammalian networks of protein interactions, signalling pathways, and transcriptional regulatory networks into BioPAX. Some of these networks were deposited into PathwayCommons, a repository for consolidating and organizing biochemical networks. The software tool Sig2BioPAX is a resource that enables experimental and computational systems biologists to contribute their identified networks and pathways of molecular interactions for integration and reuse with the rest of the research community.

  6. Uncovering a key to the process of metastasis in human cancers: a review of critical regulators of anoikis.

    PubMed

    Tan, Kevin; Goldstein, David; Crowe, Philip; Yang, Jia-Lin

    2013-11-01

    Anoikis ('homelessness' in Greek) is a form of apoptosis following the detachment of cells from the appropriate extracellular matrix (Chiarugi and Giannoni in Biochem Pharmacol 76:1352-1364, 2008). Resistance to anoikis is a critical mediator of metastasis in cancer by enabling cancer cells to survive during invasion and transport in the blood and lymph. Numerous regulators and mechanisms of anoikis in human cancer have been proposed to date. Consequently, the identification of key regulators of anoikis that can be targeted to at least partially restore anoikis sensitivity in cancer cells is important in the development of therapies to treat metastatic cancer. A literature search focusing on the regulators of anoikis in human cancer was performed on the Medline, Embase and Scopus databases. Mcl-1, Cav-1, Bcl-(xL), cFLIP, 14-3-3ζ and Bit1 appear to regulate anoikis in human cancer by participating in the intrinsic apoptotic pathway, extrinsic apoptotic pathway or caspase-independent pathways. Mcl-1, Cav-1, Bcl-(xL), cFLIP and 14-3-3ζ are suppressors of anoikis, and their upregulation confers anoikis resistance to cancer cells. Bit1 is a promoter of anoikis and is downregulated to confer anoikis resistance in metastatic cancer. Anoikis is a complex process involving the crosstalk between different signalling pathways. The dysregulated expression of key regulators of anoikis that participate in these signalling pathways promotes anoikis resistance in human cancer. These regulators of anoikis might therefore be the targets for developing therapies to overcome anoikis resistance in metastatic cancer.

  7. Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.

    PubMed Central

    Byrne, P F; McMullen, M D; Snook, M E; Musket, T A; Theuri, J M; Widstrom, N W; Wiseman, B R; Coe, E H

    1996-01-01

    Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait. PMID:11607699

  8. In Vitro Reconstitution of Metabolic Pathways: Insights into Nature’s Chemical Logic

    PubMed Central

    Lowry, Brian; Walsh, Christopher T.

    2015-01-01

    In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature’s core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway’s constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature’s biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques. PMID:26207083

  9. A thermochemical-biochemical hybrid processing of lignocellulosic biomass for producing fuels and chemicals.

    PubMed

    Shen, Yanwen; Jarboe, Laura; Brown, Robert; Wen, Zhiyou

    2015-12-01

    Thermochemical-biological hybrid processing uses thermochemical decomposition of lignocellulosic biomass to produce a variety of intermediate compounds that can be converted into fuels and chemicals through microbial fermentation. It represents a unique opportunity for biomass conversion as it mitigates some of the deficiencies of conventional biochemical (pretreatment-hydrolysis-fermentation) and thermochemical (pyrolysis or gasification) processing. Thermochemical-biological hybrid processing includes two pathways: (i) pyrolysis/pyrolytic substrate fermentation, and (ii) gasification/syngas fermentation. This paper provides a comprehensive review of these two hybrid processing pathways, including the characteristics of fermentative substrates produced in the thermochemical stage and microbial utilization of these compounds in the fermentation stage. The current challenges of these two biomass conversion pathways include toxicity of the crude pyrolytic substrates, the inhibition of raw syngas contaminants, and the mass-transfer limitations in syngas fermentation. Possible approaches for mitigating substrate toxicities are discussed. The review also provides a summary of the current efforts to commercialize hybrid processing. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. libSRES: a C library for stochastic ranking evolution strategy for parameter estimation.

    PubMed

    Ji, Xinglai; Xu, Ying

    2006-01-01

    Estimation of kinetic parameters in a biochemical pathway or network represents a common problem in systems studies of biological processes. We have implemented a C library, named libSRES, to facilitate a fast implementation of computer software for study of non-linear biochemical pathways. This library implements a (mu, lambda)-ES evolutionary optimization algorithm that uses stochastic ranking as the constraint handling technique. Considering the amount of computing time it might require to solve a parameter-estimation problem, an MPI version of libSRES is provided for parallel implementation, as well as a simple user interface. libSRES is freely available and could be used directly in any C program as a library function. We have extensively tested the performance of libSRES on various pathway parameter-estimation problems and found its performance to be satisfactory. The source code (in C) is free for academic users at http://csbl.bmb.uga.edu/~jix/science/libSRES/

  11. Searching whole genome sequences for biochemical identification features of emerging and reemerging pathogenic Corynebacterium species.

    PubMed

    Santos, André S; Ramos, Rommel T; Silva, Artur; Hirata, Raphael; Mattos-Guaraldi, Ana L; Meyer, Roberto; Azevedo, Vasco; Felicori, Liza; Pacheco, Luis G C

    2018-05-11

    Biochemical tests are traditionally used for bacterial identification at the species level in clinical microbiology laboratories. While biochemical profiles are generally efficient for the identification of the most important corynebacterial pathogen Corynebacterium diphtheriae, their ability to differentiate between biovars of this bacterium is still controversial. Besides, the unambiguous identification of emerging human pathogenic species of the genus Corynebacterium may be hampered by highly variable biochemical profiles commonly reported for these species, including Corynebacterium striatum, Corynebacterium amycolatum, Corynebacterium minutissimum, and Corynebacterium xerosis. In order to identify the genomic basis contributing for the biochemical variabilities observed in phenotypic identification methods of these bacteria, we combined a comprehensive literature review with a bioinformatics approach based on reconstruction of six specific biochemical reactions/pathways in 33 recently released whole genome sequences. We used data retrieved from curated databases (MetaCyc, PathoSystems Resource Integration Center (PATRIC), The SEED, TransportDB, UniProtKB) associated with homology searches by BLAST and profile Hidden Markov Models (HMMs) to detect enzymes participating in the various pathways and performed ab initio protein structure modeling and molecular docking to confirm specific results. We found a differential distribution among the various strains of genes that code for some important enzymes, such as beta-phosphoglucomutase and fructokinase, and also for individual components of carbohydrate transport systems, including the fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase (PTS) and the ribose-specific ATP-binging cassette (ABC) transporter. Horizontal gene transfer plays a role in the biochemical variability of the isolates, as some genes needed for sucrose fermentation were seen to be present in genomic islands. Noteworthy, using profile HMMs, we identified an enzyme with putative alpha-1,6-glycosidase activity only in some specific strains of C. diphtheriae and this may aid to understanding of the differential abilities to utilize glycogen and starch between the biovars.

  12. Pheochromocytoma-paraganglioma: Biochemical and genetic diagnosis.

    PubMed

    Cano Megías, Marta; Rodriguez Puyol, Diego; Fernández Rodríguez, Loreto; Sención Martinez, Gloria Lisette; Martínez Miguel, Patricia

    Pheochromocytomas and paragangliomas are tumours derived from neural crest cells, which can be diagnosed by biochemical measurement of metanephrine and methoxytyramine. Advances in genetic research have identified many genes involved in the pathogenesis of these tumours, suggesting that up to 35-45% may have an underlying germline mutation. These genes have a singular transcriptional signature and can be grouped into 2 clusters (or groups): cluster 1 (VHL and SHDx), involved in angiogenesis and hypoxia pathways; and cluster 2 (MEN2 and NF1), linked to the kinase signalling pathway. In turn, these genes are associated with a characteristic biochemical phenotype (noradrenergic and adrenergic), and clinical features (location, biological behaviour, age of presentation, etc.) in a large number of cases. Early diagnosis of these tumours, accompanied by a correct genetic diagnosis, should eventually become a priority to enable better treatment, early detection of complications, proper screening of family members and related tumours, as well as an improvement in the overall prognosis of these patients. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  13. Identification of biochemical adaptations in hyper- or hypocontractile hearts from phospholamban mutant mice by expression proteomics.

    PubMed

    Pan, Yan; Kislinger, Thomas; Gramolini, Anthony O; Zvaritch, Elena; Kranias, Evangelia G; MacLennan, David H; Emili, Andrew

    2004-02-24

    Phospholamban (PLN) is a critical regulator of cardiac contractility through its binding to and regulation of the activity of the sarco(endo)plasmic reticulum Ca2+ ATPase. To uncover biochemical adaptations associated with extremes of cardiac muscle contractility, we used high-throughput gel-free tandem MS to monitor differences in the relative abundance of membrane proteins in standard microsomal fractions isolated from the hearts of PLN-null mice (PLN-KO) with high contractility and from transgenic mice overexpressing a superinhibitory PLN mutant in a PLN-null background (I40A-KO) with diminished contractility. Significant differential expression was detected for a subset of the 782 proteins identified, including known membrane-associated biomarkers, components of signaling pathways, and previously uninvestigated proteins. Proteins involved in fat and carbohydrate metabolism and proteins linked to G protein-signaling pathways activating protein kinase C were enriched in I40A-KO cardiac muscle, whereas proteins linked to enhanced contractile function were enriched in PLN-KO mutant hearts. These data demonstrate that Ca2+ dysregulation, leading to elevated or depressed cardiac contractility, induces compensatory biochemical responses.

  14. Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A

    PubMed Central

    2010-01-01

    Background The archaeon, Methanosarcina acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane. Results The expression of many seemingly "redundant" genes/gene clusters establish substrate dependent control of approximately seventy genes for methane production by the pathways for methanol and acetate utilization. These include genes for soluble-type and membrane-type heterodisulfide reductases (hdr), hydrogenases including genes for a vht-type F420 non-reducing hydrogenase, molybdenum-type (fmd) as well as tungsten-type (fwd) formylmethanofuran dehydrogenases, genes for rnf and mrp-type electron transfer complexes, for acetate uptake, plus multiple genes for aha- and atp-type ATP synthesis complexes. Analysis of promoters for seven gene clusters reveal UTR leaders of 51-137 nucleotides in length, raising the possibility of both transcriptional and translational levels of control. Conclusions The above findings establish the differential and coordinated expression of two major gene families in M. acetivorans in response to carbon/energy supply. Furthermore, the quantitative mRNA measurements demonstrate the dynamic range for modulating transcript abundance. Since many of these gene clusters in M. acetivorans are also present in other Methanosarcina species including M. mazei, and in M. barkeri, these findings provide a basis for predicting related control in these environmentally significant methanogens. PMID:20178638

  15. Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

    PubMed

    Maldonado, H; Calderon, C; Burgos-Bravo, F; Kobler, O; Zuschratter, W; Ramirez, O; Härtel, S; Schneider, P; Quest, A F G; Herrera-Molina, R; Leyton, L

    2017-02-01

    Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Modeling Non-homologous End Joining

    NASA Technical Reports Server (NTRS)

    Li, Yongfeng

    2013-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed

  17. Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation

    PubMed Central

    Murabito, Ettore; Verma, Malkhey; Bekker, Martijn; Bellomo, Domenico; Westerhoff, Hans V.; Teusink, Bas; Steuer, Ralf

    2014-01-01

    Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models. PMID:25268481

  18. Proteomic analysis of proteins related to rice grain chalkiness using iTRAQ and a novel comparison system based on a notched-belly mutant with white-belly

    PubMed Central

    2014-01-01

    Background Grain chalkiness is a complex trait adversely affecting appearance and milling quality, and therefore has been one of principal targets for rice improvement. Eliminating chalkiness from rice has been a daunting task due to the complex interaction between genotype and environment and the lack of molecular markers. In addition, the molecular mechanisms underlying grain chalkiness formation are still imperfectly understood. Results We identified a notched-belly mutant (DY1102) with high percentage of white-belly, which only occurs in the bottom part proximal to the embryo. Using this mutant, a novel comparison system that can minimize the effect of genetic background and growing environment was developed. An iTRAQ-based comparative display of the proteins between the bottom chalky part and the upper translucent part of grains of DY1102 was performed. A total of 113 proteins responsible for chalkiness formation was identified. Among them, 70 proteins are up-regulated and 43 down-regulated. Approximately half of these differentially expressed proteins involved in central metabolic or regulatory pathways including carbohydrate metabolism (especially cell wall synthesis) and protein synthesis, folding and degradation, providing proteomic confirmation of the notion that chalkiness formation involves diverse but delicately regulated pathways. Protein metabolism was the most abundant category, accounting for 27.4% of the total differentially expressed proteins. In addition, down regulation of PDIL 2–3 and BiP was detected in the chalky tissue, indicating the important role of protein metabolism in grain chalkiness formation. Conclusions Using this novel comparison system, our comprehensive survey of endosperm proteomics in the notched-belly mutant provides a valuable proteomic resource for the characterization of pathways contributing to chalkiness formation at molecular and biochemical levels. PMID:24924297

  19. Systems genetics: a paradigm to improve discovery of candidate genes and mechanisms underlying complex traits.

    PubMed

    Feltus, F Alex

    2014-06-01

    Understanding the control of any trait optimally requires the detection of causal genes, gene interaction, and mechanism of action to discover and model the biochemical pathways underlying the expressed phenotype. Functional genomics techniques, including RNA expression profiling via microarray and high-throughput DNA sequencing, allow for the precise genome localization of biological information. Powerful genetic approaches, including quantitative trait locus (QTL) and genome-wide association study mapping, link phenotype with genome positions, yet genetics is less precise in localizing the relevant mechanistic information encoded in DNA. The coupling of salient functional genomic signals with genetically mapped positions is an appealing approach to discover meaningful gene-phenotype relationships. Techniques used to define this genetic-genomic convergence comprise the field of systems genetics. This short review will address an application of systems genetics where RNA profiles are associated with genetically mapped genome positions of individual genes (eQTL mapping) or as gene sets (co-expression network modules). Both approaches can be applied for knowledge independent selection of candidate genes (and possible control mechanisms) underlying complex traits where multiple, likely unlinked, genomic regions might control specific complex traits. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides.

    PubMed

    Allred, Benjamin E; Rupert, Peter B; Gauny, Stacey S; An, Dahlia D; Ralston, Corie Y; Sturzbecher-Hoehne, Manuel; Strong, Roland K; Abergel, Rebecca J

    2015-08-18

    Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.

  1. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    PubMed

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  2. Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides

    PubMed Central

    Allred, Benjamin E.; Rupert, Peter B.; Gauny, Stacey S.; An, Dahlia D.; Ralston, Corie Y.; Sturzbecher-Hoehne, Manuel; Strong, Roland K.; Abergel, Rebecca J.

    2015-01-01

    Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin–transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein–ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications. PMID:26240330

  3. Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

    PubMed Central

    Ahn, Hyo-Min; Koh, Young Ho

    2016-01-01

    We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved. PMID:27313903

  4. Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway*

    PubMed Central

    Barta, Michael L.; Thomas, Keisha; Yuan, Hongling; Lovell, Scott; Battaile, Kevin P.; Schramm, Vern L.; Hefty, P. Scott

    2014-01-01

    The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.g. menaquionone or ubiquinone). The sources or biosynthetic pathways required to obtain these electron carriers within C. trachomatis are poorly understood. The 1.58Å crystal structure of C. trachomatis hypothetical protein CT263 presented here supports a role in quinone biosynthesis. Although CT263 lacks sequence-based functional annotation, the crystal structure of CT263 displays striking structural similarity to 5′-methylthioadenosine nucleosidase (MTAN) enzymes. Although CT263 lacks the active site-associated dimer interface found in prototypical MTANs, co-crystal structures with product (adenine) or substrate (5′-methylthioadenosine) indicate that the canonical active site residues are conserved. Enzymatic characterization of CT263 indicates that the futalosine pathway intermediate 6-amino-6-deoxyfutalosine (kcat/Km = 1.8 × 103 m−1 s−1), but not the prototypical MTAN substrates (e.g. S-adenosylhomocysteine and 5′-methylthioadenosine), is hydrolyzed. Bioinformatic analyses of the chlamydial proteome also support the futalosine pathway toward the synthesis of menaquinone in Chlamydiaceae. This report provides the first experimental support for quinone synthesis in Chlamydia. Menaquinone synthesis provides another target for agents to combat C. trachomatis infection. PMID:25253688

  5. A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

    PubMed Central

    Lucarelli, Marco; Bruno, Sabina Maria; Pierandrei, Silvia; Ferraguti, Giampiero; Stamato, Antonella; Narzi, Fabiana; Amato, Annalisa; Cimino, Giuseppe; Bertasi, Serenella; Quattrucci, Serena; Strom, Roberto

    2015-01-01

    Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway. PMID:25910067

  6. Interactions of C4 subtype metabolic activities and transport in maize are revealed through the characterization of DCT2 mutants

    USDA-ARS?s Scientific Manuscript database

    C4 photosynthesis is an elaborate set of metabolic pathways that utilize specialized anatomical and biochemical adaptations to concentrate CO2 around RuBisCO. The activities of the C4 pathways are coordinated between two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), and rely hea...

  7. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS

    EPA Science Inventory

    Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis ...

  8. Haptoglobin, hemopexin, and related defense pathways-basic science, clinical perspectives, and drug development.

    PubMed

    Schaer, Dominik J; Vinchi, Francesca; Ingoglia, Giada; Tolosano, Emanuela; Buehler, Paul W

    2014-01-01

    Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 × 10(6) RBCs/second. Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions.

  9. Earthworms and nutrient availability: the ecosystem engineer as (bio)chemical engineer

    NASA Astrophysics Data System (ADS)

    van Groenigen, Jan Willem; Ros, Mart; Vos, Hannah; De Deyn, Gerlinde; Hiemstra, Tjisse; Oenema, Oene; Koopmans, Gerwin

    2017-04-01

    The ability of earthworms to increase plant production has long been recognized. However, the pathways through which they do so, and the magnitude of this effect, have not been conclusively addressed. In two studies we address these issues for nitrogen (N) and phosphorus (P) availability to plants. In the first study, a meta-analysis, we concluded that earthworm presence increases crop yield on average with 26% and aboveground biomass with 24%. The positive effects of earthworms increase when more residue is returned to the soil, but disappear when soil N availability is high. This suggests that earthworms stimulate plant growth predominantly through N mineralization from soil organic matter or crop residue. In a second study, we tested the effect of earthworms on plant P uptake from inorganic sources. In a greenhouse experiment on a soil with low P availability we showed that presence of the anecic earthworm Lumbricus terrestris resulted in increased aboveground biomass (from 164 to 188 g dry matter m-2) and P uptake (from 0.21 to 0.27 g m-2). Concentrations of total dissolved P and dissolved inorganic P in water extractions of earthworm casts were 7-9 times higher than in those of bulk soil. Using advanced surface complexation modelling, we showed that these effects were primarily related to desorption of inorganic P due to competition with organic carbon for binding sites. We conclude that earthworms can alter nutrient cycling and increase N and P uptake by plants through a combination of biochemical and chemical pathways. Earthworms are most likely to stimulate N uptake in organic farming systems and tropical subsistence farming, which largely rely on nutrient mineralization. Additional benefits of earthworms might be expected in conventional farming systems with low levels of available P.

  10. Modulation of nitrergic signalling pathway by American ginseng attenuates chronic unpredictable stress-induced cognitive impairment, neuroinflammation, and biochemical alterations.

    PubMed

    Rinwa, Puneet; Kumar, Anil

    2014-02-01

    Prolonged stress causes extensive loss of neurons leading to deficits in cognitive performance. Increasing evidence indicates that accumulation of intercellular messenger, nitric oxide (NO), plays a crucial role in the pathogenesis of memory disorders. American ginseng (AG) is known to show protection in different animal models of neurological diseases; however, its exact mechanism of action is not clearly understood. Therefore, the current study was designed to investigate the interaction of AG against chronic unpredictable stress (CUS)-associated behavioral and biochemical alterations and the probable role of nitrergic pathway in this effect. Male Laca mice were exposed to a series of stressors along with drug/vehicle treatment daily for 28 days. CUS paradigm caused significant impairment in both acquisition and retention memory as measured in Morris water maze and elevated plus maze task. This was coupled with alterations in oxidative stress markers, mitochondrial enzyme complex activities, pro-inflammatory cytokine (TNF-α), and acetylcholinesterase levels in the hippocampus as compared with naïve group. Besides, there was a marked increase in serum corticosterone levels. AG (100, 200 mg/kg; p.o.) treatment significantly improved cognitive impairment; reduced TNF-α, acetylcholinesterase, and corticosterone levels; and attenuated oxidative-nitrergic stress. Furthermore, pre-treatment of L-arginine (100 mg/kg; i.p.), a nitric oxide donor, with subeffective dose of AG (100 mg/kg; p.o.) reversed its protective effects. However, L-NAME (10 mg/kg, i.p.), a non-specific NO synthase inhibitor, potentiated the effects of AG. Our findings suggest that modulation of nitrergic signalling cascade is involved in the protective effects of AG against CUS-induced cognitive dysfunction, oxidative stress, and neuroinflammation.

  11. Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges.

    PubMed

    Zhang, Yi-Heng Percival

    2015-11-15

    The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Chemical modulation of glycerolipid signaling and metabolic pathways

    PubMed Central

    Scott, Sarah A.; Mathews, Thomas P.; Ivanova, Pavlina T.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    Thirty years ago, glycerolipids captured the attention of biochemical researchers as novel cellular signaling entities. We now recognize that these biomolecules occupy signaling nodes critical to a number of physiological and pathological processes. Thus, glycerolipid-metabolizing enzymes present attractive targets for new therapies. A number of fields—ranging from neuroscience and cancer to diabetes and obesity—have elucidated the signaling properties of glycerolipids. The biochemical literature teems with newly emerging small molecule inhibitors capable of manipulating glycerolipid metabolism and signaling. This ever-expanding pool of chemical modulators appears daunting to those interested in exploiting glycerolipid-signaling pathways in their model system of choice. This review distills the current body of literature surrounding glycerolipid metabolism into a more approachable format, facilitating the application of small molecule inhibitors to novel systems. PMID:24440821

  13. Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure

    PubMed Central

    Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

  14. Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

    PubMed

    Garrocho-Villegas, Verónica; Aguilar C, Raúl; Sánchez de Jiménez, Estela

    2013-12-23

    The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

  15. Harnessing Intracellular Biochemical Pathways for In Vitro Synthesis of Designer Tellurium Nanorods.

    PubMed

    Xiong, Ling-Hong; Cui, Ran; Zhang, Zhi-Ling; Tu, Jia-Wei; Shi, Yun-Bo; Pang, Dai-Wen

    2015-10-28

    Synthesizing nanomaterials of desired properties is a big challenge, which requires extremely harsh conditions and/or use of toxic materials. More recently developed in vivo methods have brought a different set of problems such as separation and purification of nanomaterials made in vivo. Here, a novel approach that harnesses cellular pathways for in vitro synthesis of high-quality tellurium nanorods with tunable lengths and optical properties is reported. It is first demonstrated that in vivo biochemical pathways could be used to synthesize Te nanorods via the intracellular reduction of TeO3(2-) in living Staphylococcus aureus cells. The pathways to set up a quasi-biological system for Te precursor formation are then utilized, which could further synthesize Te nanorods in vitro. This allows to successfully synthesize in vitro, under routine laboratory conditions, Te nanorods with uniform and tunable lengths, ranging from about 10 to 200 nm, and controllable optical properties with high molar extinction coefficients. The approach here should open new avenues for controllable, facile, and efficient synthesis of designer nanomaterials for diverse industrial and biomedical applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. The Recycling Endosome of Madin-Darby Canine Kidney Cells Is a Mildly Acidic Compartment Rich in Raft Components

    PubMed Central

    Gagescu, Raluca; Demaurex, Nicolas; Parton, Robert G.; Hunziker, Walter; Huber, Lukas A.; Gruenberg, Jean

    2000-01-01

    We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. PMID:10930469

  17. Molecular Pathways

    PubMed Central

    Lok, Benjamin H.; Powell, Simon N.

    2012-01-01

    The Rad52 protein was largely ignored in humans and other mammals when the mouse knockout revealed a largely “no-effect” phenotype. However, using synthetic lethal approaches to investigate context dependent function, new studies have shown that Rad52 plays a key survival role in cells lacking the function of the BRCA1-BRCA2 pathway of homologous recombination. Biochemical studies also showed significant differences between yeast and human Rad52, in which yeast Rad52 can promote strand invasion of RPA-coated single-stranded DNA in the presence of Rad51, but human Rad52 cannot. This results in the paradox of how is human Rad52 providing Rad51 function: presumably there is something missing in the biochemical assays that exists in-vivo, but the nature of this missing factor is currently unknown. Recent studies have suggested that Rad52 provides back-up Rad51 function for all members of the BRCA1-BRCA2 pathway, suggesting that Rad52 may be a target for therapy in BRCA pathway deficient cancers. Screening for ways to inhibit Rad52 would potentially provide a complementary strategy for targeting BRCA-deficient cancers in addition to PARP inhibitors. PMID:23071261

  18. Complex Degradation Processes Lead to Non-Exponential Decay Patterns and Age-Dependent Decay Rates of Messenger RNA

    PubMed Central

    Deneke, Carlus; Lipowsky, Reinhard; Valleriani, Angelo

    2013-01-01

    Experimental studies on mRNA stability have established several, qualitatively distinct decay patterns for the amount of mRNA within the living cell. Furthermore, a variety of different and complex biochemical pathways for mRNA degradation have been identified. The central aim of this paper is to bring together both the experimental evidence about the decay patterns and the biochemical knowledge about the multi-step nature of mRNA degradation in a coherent mathematical theory. We first introduce a mathematical relationship between the mRNA decay pattern and the lifetime distribution of individual mRNA molecules. This relationship reveals that the mRNA decay patterns at steady state expression level must obey a general convexity condition, which applies to any degradation mechanism. Next, we develop a theory, formulated as a Markov chain model, that recapitulates some aspects of the multi-step nature of mRNA degradation. We apply our theory to experimental data for yeast and explicitly derive the lifetime distribution of the corresponding mRNAs. Thereby, we show how to extract single-molecule properties of an mRNA, such as the age-dependent decay rate and the residual lifetime. Finally, we analyze the decay patterns of the whole translatome of yeast cells and show that yeast mRNAs can be grouped into three broad classes that exhibit three distinct decay patterns. This paper provides both a method to accurately analyze non-exponential mRNA decay patterns and a tool to validate different models of degradation using decay data. PMID:23408982

  19. Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate : new insights from structural and biochemical studies on human RPE.

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

    Liang, W.; Ouyang, S.; Shaw, N.

    2011-02-01

    The pentose phosphate pathway (PPP) confers protection against oxidative stress by supplying NADPH necessary for the regeneration of glutathione, which detoxifies H{sub 2}O{sub 2} into H{sub 2}O and O{sub 2}. RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} for catalysis. Structures of the binary complexes of hRPE with D-ribulose 5-phosphate and D-xylulose 5-phosphate provide the first detailed molecular insights into the binding mode ofmore » physiological ligands and reveal an octahedrally coordinated Fe{sup 2+} ion buried deep inside the active site. Human RPE folds into a typical ({beta}/{alpha}){sub 8} triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. Two aspartic acids are well positioned to carry out the proton transfers in an acid-base type of reaction mechanism. Interestingly, mutating Ser-10 to alanine almost abolished the enzymatic activity, while L12A and M72A mutations resulted in an almost 50% decrease in the activity. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.« less

  20. Brain banks as key part of biochemical and molecular studies on cerebral cortex involvement in Parkinson's disease.

    PubMed

    Ravid, Rivka; Ferrer, Isidro

    2012-04-01

    Exciting developments in basic and clinical neuroscience and recent progress in the field of Parkinson's disease (PD) are partly a result of the availability of human specimens obtained through brain banks. These banks have optimized the methodological, managerial and organizational procedures; standard operating procedures; and ethical, legal and social issues, including the code of conduct for 21st Century brain banking and novel protocols. The present minireview focuses on current brain banking organization and management, as well as the likely future direction of the brain banking field. We emphasize the potentials and pitfalls when using high-quality specimens of the human central nervous system for advancing PD research. PD is a generalized disease in which α-synuclein is not a unique component but, instead, is only one of the players accounting for the complex impairment of biochemical/molecular processes involved in metabolic pathways. This is particularly important in the cerebral cortex, where altered cognition has a complex neurochemical substrate. Mitochondria and energy metabolism impairment, abnormal RNA, microRNA, protein synthesis, post-translational protein modifications and alterations in the lipid composition of membranes and lipid rafts are part of these complementary factors. We have to be alert to the possible pitfalls of each specimen and its suitability for a particular study. Not all samples qualify for the study of DNA, RNA, proteins, post-translational modifications, lipids and metabolomes, although the use of carefully selected samples and appropriate methods minimizes pitfalls and errors and guarantees high-quality reserach. © 2012 The Authors Journal compilation © 2012 FEBS.

  1. Mapping complex traits as a dynamic system

    PubMed Central

    Sun, Lidan; Wu, Rongling

    2017-01-01

    Despite increasing emphasis on the genetic study of quantitative traits, we are still far from being able to chart a clear picture of their genetic architecture, given an inherent complexity involved in trait formation. A competing theory for studying such complex traits has emerged by viewing their phenotypic formation as a “system” in which a high-dimensional group of interconnected components act and interact across different levels of biological organization from molecules through cells to whole organisms. This system is initiated by a machinery of DNA sequences that regulate a cascade of biochemical pathways to synthesize endophenotypes and further assemble these endophenotypes toward the end-point phenotype in virtue of various developmental changes. This review focuses on a conceptual framework for genetic mapping of complex traits by which to delineate the underlying components, interactions and mechanisms that govern the system according to biological principles and understand how these components function synergistically under the control of quantitative trait loci (QTLs) to comprise a unified whole. This framework is built by a system of differential equations that quantifies how alterations of different components lead to the global change of trait development and function, and provides a quantitative and testable platform for assessing the multiscale interplay between QTLs and development. The method will enable geneticists to shed light on the genetic complexity of any biological system and predict, alter or engineer its physiological and pathological states. PMID:25772476

  2. Two distinct arsenite-resistant variants of Leishmania amazonensis take different routes to achieve resistance as revealed by comparative transcriptomics.

    PubMed

    Lin, Yi-Chun; Hsu, Ju-Yu; Shu, Jui-Hsu; Chi, Yi; Chiang, Su-Chi; Lee, Sho Tone

    2008-11-01

    Genome-wide search for the genes involved in arsenite resistance in two distinct variants A and A' of Leishmania amazonensis revealed that the two variants used two different mechanisms to achieve resistance, even though these two variants were derived from the same clone and selected against arsenite under the same conditions. In variant A, the variant with DNA amplification, the biochemical pathways for detoxification of oxidative stress, the energy generation system to support the biochemical and physiological needs of the variant for DNA and protein synthesis and the arsenite translocating system to dispose arsenite are among the primary biochemical events that are upregulated under the arsenite stress to gain resistance. In variant A', the variant without DNA amplification, the upregulation of aquaglyceroporin (AQP) gene and the high level of resistance to arsenate point to the direction that the resistance gained by the variant is due to arsenate which is probably oxidized from arsenite in the arsenite solution used for selection and the maintenance of the cell culture. As a result of the AQP upregulation for arsenite disposal, a different set of biochemical pathways for detoxification of oxidative stress, energy generation and cellular signaling are upregulated to sustain the growth of the variant to gain resistance to arsenate. From current evidences, reactive oxygen species (ROS) overproduced by the parasite soon after exposure to arsenite appear to play an instrumental role in both variants to initiate the subsequent biochemical events that allow the same clone of L. amazonensis to take two totally different routes to diverge into two different variants.

  3. Microfluidics meets metabolomics to reveal the impact of Campylobacter jejuni infection on biochemical pathways.

    PubMed

    Mortensen, Ninell P; Mercier, Kelly A; McRitchie, Susan; Cavallo, Tammy B; Pathmasiri, Wimal; Stewart, Delisha; Sumner, Susan J

    2016-06-01

    Microfluidic devices that are currently being used in pharmaceutical research also have a significant potential for utilization in investigating exposure to infectious agents. We have established a microfluidic device cultured with Caco-2 cells, and utilized metabolomics to investigate the biochemical responses to the bacterial pathogen Campylobacter jejuni. In the microfluidic devices, Caco-2 cells polarize at day 5, are uniform, have defined brush borders and tight junctions, and form a mucus layer. Metabolomics analysis of cell culture media collected from both Caco-2 cell culture systems demonstrated a more metabolic homogenous biochemical profile in the media collected from microfluidic devices, compared with media collected from transwells. GeneGo pathway mapping indicated that aminoacyl-tRNA biosynthesis was perturbed by fluid flow, suggesting that fluid dynamics and shear stress impacts the cells translational quality control. Both microfluidic device and transwell culturing systems were used to investigate the impact of Campylobacter jejuni infection on biochemical processes. Caco-2 cells cultured in either system were infected at day 5 with C. jejuni 81-176 for 48 h. Metabolomics analysis clearly differentiated C. jejuni 81-176 infected and non-infected medias collected from the microfluidic devices, and demonstrated that C. jejuni 81-176 infection in microfluidic devices impacts branched-chain amino acid metabolism, glycolysis, and gluconeogenesis. In contrast, no distinction was seen in the biochemical profiles of infected versus non-infected media collected from cells cultured in transwells. Microfluidic culturing conditions demonstrated a more metabolically homogenous cell population, and present the opportunity for studying host-pathogen interactions for extended periods of time.

  4. Microfluidics Meets Metabolomics to Reveal the Impact of Campylobacter jejuni Infection on Biochemical Pathways

    PubMed Central

    Mortensen, Ninell P.; Mercier, Kelly A.; McRitchie, Susan; Cavallo, Tammy B.; Pathmasiri, Wimal; Stewart, Delisha; Sumner, Susan J.

    2016-01-01

    Microfluidic devices that are currently being used in pharmaceutical research also have a significant potential for utilization in investigating exposure to infectious agents. We have established a microfluidic device cultured with Caco-2 cells, and utilized metabolomics to investigate the biochemical responses to the bacterial pathogen Campylobacter jejuni. In the microfluidic devices, Caco-2 cells polarize at day 5, are uniform, have defined brush borders and tight junctions, and form a mucus layer. Metabolomics analysis of cell culture media collected from both Caco-2 cell culture systems demonstrated a more metabolic homogenous biochemical profile in the media collected from microfluidic devices, compared with media collected from transwells. GeneGo pathway mapping indicated that aminoacyl-tRNA biosynthesis was perturbed by fluid flow, suggesting that fluid dynamics and shear stress impacts the cells translational quality control. Both microfluidic device and transwell culturing systems were used to investigate the impact of Campylobacter jejuni infection on biochemical processes. Caco-2 cells cultured in either system were infected at day 5 with C. jejuni 81-176 for 48 hours. Metabolomics analysis clearly differentiated C. jejuni 81-176 infected and non-infected medias collected from the microfluidic devices, and demonstrated that C. jejuni 81-176 infection in microfluidic devices impacts branched-chain amino acid metabolism, glycolysis, and gluconeogenesis. In contrast, no distinction was seen in the biochemical profiles of infected versus non-infected media collected from cells cultured in transwells. Microfluidic culturing conditions demonstrated a more metabolically homogenous cell population, and present the opportunity for studying host-pathogen interactions for extended periods of time. PMID:27231016

  5. Metabologenomics of Phaeochromocytoma and Paraganglioma: An Integrated Approach for Personalised Biochemical and Genetic Testing

    PubMed Central

    Eisenhofer, Graeme; Klink, Barbara; Richter, Susan; Lenders, Jacques WM; Robledo, Mercedes

    2017-01-01

    The tremendous advances over the past two decades in both clinical genetics and biochemical testing of chromaffin cell tumours have led to new considerations about how these aspects of laboratory medicine can be integrated to improve diagnosis and management of affected patients. With germline mutations in 15 genes now identified to be responsible for over a third of all cases of phaeochromocytomas and paragangliomas, these tumours are recognised to have one of the richest hereditary backgrounds among all neoplasms. Depending on the mutation, tumours show distinct differences in metabolic pathways that relate to or even directly impact clinical presentation. At the same time, there has been improved understanding about how catecholamines are synthesised, stored, secreted and metabolised by chromaffin cell tumours. Although the tumours may not always secrete catecholamines it has become clear that almost all continuously produce and metabolise catecholamines. This has not only fuelled changes in laboratory medicine, but has also assisted in recognition of genotype-biochemical phenotype relationships important for diagnostics and clinical care. In particular, differences in catecholamine and energy pathway metabolomes can guide genetic testing, assist with test interpretation and provide predictions about the nature, behaviour and imaging characteristics of the tumours. Conversely, results of genetic testing are important for guiding how routine biochemical testing should be employed and interpreted in surveillance programmes for at-risk patients. In these ways there are emerging needs for modern laboratory medicine to seamlessly integrate biochemical and genetic testing into the diagnosis and management of patients with chromaffin cell tumours. PMID:29332973

  6. Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

    PubMed

    Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES.

  7. Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

    PubMed Central

    Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES. PMID:22465851

  8. Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

    PubMed Central

    Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

    2015-01-01

    Background and Aims The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Methods Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. Key Results In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. Conclusions These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III. PMID:26292995

  9. A longitudinal analysis of the effects of age on the blood plasma metabolome in the common marmoset, Callithrix jacchus

    PubMed Central

    Hoffman, Jessica M.; Tran, ViLinh; Wachtman, Lynn M.; Green, Cara L.; Jones, Dean P.; Promislow, Daniel E.L.

    2016-01-01

    Primates tend to be long-lived for their size with humans being the longest lived of all primates. There are compelling reasons to understand the underlying age-related processes that shape human lifespan. But the very fact of our long lifespan that makes it so compelling, also makes it especially difficult to study. Thus, in studies of aging, researchers have turned to non-human primate models, including chimpanzees, baboons, and rhesus macaques. More recently, the common marmoset, Callithrix jacchus, has been recognized as a particularly valuable model in studies of aging, given its small size, ease of housing in captivity, and relatively short lifespan. However, little is known about the physiological changes that occur as marmosets age. To begin to fill in this gap, we utilized high sensitivity metabolomics to define the longitudinal biochemical changes associated with age in the common marmoset. We measured 2104 metabolites from blood plasma at three separate time points over a 17-month period, and we completed both a cross-sectional and longitudinal analysis of the metabolome. We discovered hundreds of metabolites associated with age and body weight in both male and female animals. Our longitudinal analysis identified age-associated metabolic pathways that were not found in our cross-sectional analysis. Pathways enriched for age-associated metabolites included tryptophan, nucleotide, and xenobiotic metabolism, suggesting these biochemical pathways might play an important role in the basic mechanisms of aging in primates. Moreover, we found that many metabolic pathways associated with age were sex specific. Our work illustrates the power of longitudinal approaches, even in a short time frame, to discover novel biochemical changes that occur with age. PMID:26805607

  10. Genetic and Environmental Pathways in Type 1 Diabetes Complication

    DTIC Science & Technology

    2008-06-01

    obese diabetic mice. Biochem Biophys Res Commun 2002. 294:592-596. 4. Beyan H, Goodier MR, Nawroly NS, Hawa MI, Bustin SA, Ogunkolade WB, Londei M...peroxidation in patients with hyperglycemic crisis . Diabetes 2004. 53:2079-2086. 14 In our second quarterly scientific progress report (09/01/07 – 11...cells induced to differentiate into insulin-positive cells. Biochem Biophys Res Commun . 2007;357(2):414-20. 44. Takahashi K, Tanabe K, Ohnuki M, Narita

  11. Timescale analysis of rule-based biochemical reaction networks

    PubMed Central

    Klinke, David J.; Finley, Stacey D.

    2012-01-01

    The flow of information within a cell is governed by a series of protein-protein interactions that can be described as a reaction network. Mathematical models of biochemical reaction networks can be constructed by repetitively applying specific rules that define how reactants interact and what new species are formed upon reaction. To aid in understanding the underlying biochemistry, timescale analysis is one method developed to prune the size of the reaction network. In this work, we extend the methods associated with timescale analysis to reaction rules instead of the species contained within the network. To illustrate this approach, we applied timescale analysis to a simple receptor-ligand binding model and a rule-based model of Interleukin-12 (IL-12) signaling in näive CD4+ T cells. The IL-12 signaling pathway includes multiple protein-protein interactions that collectively transmit information; however, the level of mechanistic detail sufficient to capture the observed dynamics has not been justified based upon the available data. The analysis correctly predicted that reactions associated with JAK2 and TYK2 binding to their corresponding receptor exist at a pseudo-equilibrium. In contrast, reactions associated with ligand binding and receptor turnover regulate cellular response to IL-12. An empirical Bayesian approach was used to estimate the uncertainty in the timescales. This approach complements existing rank- and flux-based methods that can be used to interrogate complex reaction networks. Ultimately, timescale analysis of rule-based models is a computational tool that can be used to reveal the biochemical steps that regulate signaling dynamics. PMID:21954150

  12. An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: Structural, biochemical and gene expression analysis

    PubMed Central

    Chim, Nicholas; Riley, Robert; The, Juliana; Im, Soyeon; Segelke, Brent; Lekin, Tim; Yu, Minmin; Hung, Li Wei; Terwilliger, Tom; Whitelegge, Julian P.; Goulding, Celia W.

    2010-01-01

    Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here we present structural, biochemical and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pKa for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein co-expression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicate anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions. PMID:20060836

  13. Biochemical analysis of force-sensitive responses using a large-scale cell stretch device.

    PubMed

    Renner, Derrick J; Ewald, Makena L; Kim, Timothy; Yamada, Soichiro

    2017-09-03

    Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.

  14. Complexity VIII. Ontology of closure in complex systems: The C* hypothesis and the O° notation

    NASA Astrophysics Data System (ADS)

    Chandler, Jerry LR

    1999-03-01

    Closure is a common characteristic of mathematical, natural and socio-cultural systems. Whether one is describing a graph, a molecule, a cell, a human, or a nation state, closure is implicitly understood. An objective of this paper is to continue a construction of a systematic framework for closure which is sufficient for future quantitative transdisciplinary investigations. A further objective is to extend the Birkhoff-von Neumann criterion for quantum systems to complex natural objects. The C* hypothesis is being constructed to be consistent with algebraic category theory (Ehresmann and Vanbremeersch, 1987, 1997, Chandler, 1990, 1991, Chandler, Ehresmann and Vanbremeersch, 1996). Five aspects of closure will be used to construct a framework for categories of complex systems: 1. Truth functions in mathematics and the natural sciences 2. Systematic descriptions in the mks and O° notations 3. Organizational structures in hierarchical scientific languages 4. Transitive organizational pathways in the causal structures of complex behaviors 5. Composing additive, multiplicative and exponential operations in complex systems Truth functions can be formal or objective or subjective, depending on the complexity of the system and on our capability to represent the fine structure of the system symbolically, observationally or descriptively. "Complete" material representations of the fine structure of a system may allow truth functions to be created over sets of one to one correspondences. Less complete descriptions can support less stringent truth functions based on coherence or subjective judgments. The role of human values in creating and perpetuating truth functions can be placed in context of the degree of fine structure in the system's description. The organization of complex systems are hypothesized to be categorizable into degrees relative to one another, thereby creating an ordering relationship. This ordering relationship is denoted by the symbols: O°1, O°2,O°3... For example, for material systems, an ordering relation such as particles, atoms, molecules, cells, tissues, organs, individuals and social groups might be assigned to classify observations for medical purposes. The C* hypothesis asserts that any complex system can be described in terms of four enumerable concepts: closure, conformation, concatenation and cyclicity. Mappings between objects are constructed within a notation for organization. Causality is organized within C* as pathways of relationships in time. The notation of organizational degrees is used to distinguish a directionality for causality: 1. bottom-up (energy flows) 2. top-down (control processes or dominating variables), 3. outside — inward (ecoment on organism) and 4. inside — outward (organism on ecoment). Closures are asserted to emerge from evolutionary cooperation. It is asserted that truth functions emerged from the necessity of an organism to identify ecoments where life can prosper. For example, basic truth functions of mathematics (operations of addition, multiplication and exponentiation) are made operationally consistent within the biochemical operations of sustaining exponential cellular growth. These fundamental mathematical functions can provide a logical basis (in conjunction with conservation rules) for a construction of complex material categories at higher degrees of organization. It is remarked that these simple functions suggests a biochemical origin for the intuitionistic philosophy of mathematics. The emergence and success of mathematics is conjectured to result from the need to acquire a consistent basis for communication among individuals seeking to cooperate socially. This suggests a cultural closure over a collection of individual closures.

  15. PathwayAccess: CellDesigner plugins for pathway databases.

    PubMed

    Van Hemert, John L; Dickerson, Julie A

    2010-09-15

    CellDesigner provides a user-friendly interface for graphical biochemical pathway description. Many pathway databases are not directly exportable to CellDesigner models. PathwayAccess is an extensible suite of CellDesigner plugins, which connect CellDesigner directly to pathway databases using respective Java application programming interfaces. The process is streamlined for creating new PathwayAccess plugins for specific pathway databases. Three PathwayAccess plugins, MetNetAccess, BioCycAccess and ReactomeAccess, directly connect CellDesigner to the pathway databases MetNetDB, BioCyc and Reactome. PathwayAccess plugins enable CellDesigner users to expose pathway data to analytical CellDesigner functions, curate their pathway databases and visually integrate pathway data from different databases using standard Systems Biology Markup Language and Systems Biology Graphical Notation. Implemented in Java, PathwayAccess plugins run with CellDesigner version 4.0.1 and were tested on Ubuntu Linux, Windows XP and 7, and MacOSX. Source code, binaries, documentation and video walkthroughs are freely available at http://vrac.iastate.edu/~jlv.

  16. Modeling cellular compartmentation in one-carbon metabolism

    PubMed Central

    Scotti, Marco; Stella, Lorenzo; Shearer, Emily J.; Stover, Patrick J.

    2015-01-01

    Folate-mediated one-carbon metabolism (FOCM) is associated with risk for numerous pathological states including birth defects, cancers, and chronic diseases. Although the enzymes that constitute the biological pathways have been well described and their interdependency through the shared use of folate cofactors appreciated, the biological mechanisms underlying disease etiologies remain elusive. The FOCM network is highly sensitive to nutritional status of several B-vitamins and numerous penetrant gene variants that alter network outputs, but current computational approaches do not fully capture the dynamics and stochastic noise of the system. Combining the stochastic approach with a rule-based representation will help model the intrinsic noise displayed by FOCM, address the limited flexibility of standard simulation methods for coarse-graining the FOCM-associated biochemical processes, and manage the combinatorial complexity emerging from reactions within FOCM that would otherwise be intractable. PMID:23408533

  17. Unveiling the functional diversity of the alpha/beta hydrolase superfamily in the plant kingdom.

    PubMed

    Mindrebo, Jeffrey T; Nartey, Charisse M; Seto, Yoshiya; Burkart, Michael D; Noel, Joseph P

    2016-12-01

    The alpha/beta hydrolase (ABH) superfamily is a widespread and functionally malleable protein fold recognized for its diverse biochemical activities across all three domains of life. ABH enzymes possess unexpected catalytic activity in the green plant lineage through selective alterations in active site architecture and chemistry. Furthermore, the ABH fold serves as the core structure for phytohormone and ligand receptors in the gibberellin, strigolactone, and karrikin signaling pathways in plants. Despite recent discoveries, the ABH family is sparsely characterized in plants, a sessile kingdom known to evolve complex and specialized chemical adaptations as survival responses to widely varying biotic and abiotic ecologies. This review calls attention to the ABH superfamily in the plant kingdom to highlight the functional adaptability of the ABH fold. Copyright © 2016. Published by Elsevier Ltd.

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

    Garige, Mamatha; Walters, Eric, E-mail: ewalters@howard.edu

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstAmore » gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.« less

  19. Selective disruption of the AKAP signaling complexes.

    PubMed

    Kennedy, Eileen J; Scott, John D

    2015-01-01

    Synthesis of the second messenger cAMP activates a variety of signaling pathways critical for all facets of intracellular regulation. Protein kinase A (PKA) is the major cAMP-responsive effector. Where and when this enzyme is activated has profound implications on the cellular role of PKA. A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by orchestrating spatial and temporal aspects of PKA action. A popular means of evaluating the impact of these anchored signaling events is to biochemically interfere with the PKA-AKAP interface. Hence, peptide disruptors of PKA anchoring are valuable tools in the investigation of local PKA action. This article outlines the development of PKA isoform-selective disruptor peptides, documents the optimization of cell-soluble peptide derivatives, and introduces alternative cell-based approaches that interrogate other aspects of the PKA-AKAP interface.

  20. Plant-bacterial pathogen interactions mediated by type III effectors.

    PubMed

    Feng, Feng; Zhou, Jian-Min

    2012-08-01

    Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. [Implications of the new etiophatogenic approach in the classification of constitutional and genetic bone diseases].

    PubMed

    Morales Piga, Antonio; Alonso Ferreira, Verónica; Villaverde-Hueso, Ana

    2011-01-01

    Recent years have seen an unprecedented increase in the knowledge and understanding of biochemical disturbances involved on constitutional bone disorders. Recognition of the genetic background as the common cause of these diseases prompted the substitution of the term «constitutional» by «genetic», in referring to them. Understanding physiopathological bases by finding out the altered metabolic pathways as well as their regulatory and control systems, favours an earlier and more accurate diagnosis based on interdisciplinary collaboration. Although clinical and radiological assessment remains crucial in the study of these disorders, ever more often the diagnosis is achieved by molecular and genetic analysis. Elucidation of the damaged underlying molecular mechanisms offers targets potentially useful for therapeutic research in these complex and often disabling diseases. 2010 Elsevier España, S.L. All rights reserved.

  2. Biochemical and Functional Interactions of Human Papillomavirus Proteins with Polycomb Group Proteins

    PubMed Central

    McLaughlin-Drubin, Margaret E.; Munger, Karl

    2013-01-01

    The role of enzymes involved in polycomb repression of gene transcription has been studied extensively in human cancer. Polycomb repressive complexes mediate oncogene-induced senescence, a principal innate cell-intrinsic tumor suppressor pathway that thwarts expansion of cells that have suffered oncogenic hits. Infections with human cancer viruses including human papillomaviruses (HPVs) and Epstein-Barr virus can trigger oncogene-induced senescence, and the viruses have evolved strategies to abrogate this response in order to establish an infection and reprogram their host cells to establish a long-term persistent infection. As a consequence of inhibiting polycomb repression and evading oncogene induced-senescence, HPV infected cells have an altered epigenetic program as evidenced by aberrant homeobox gene expression. Similar alterations are frequently observed in non-virus associated human cancers and may be harnessed for diagnosis and therapy. PMID:23673719

  3. MetaMapp: mapping and visualizing metabolomic data by integrating information from biochemical pathways and chemical and mass spectral similarity

    PubMed Central

    2012-01-01

    Background Exposure to environmental tobacco smoke (ETS) leads to higher rates of pulmonary diseases and infections in children. To study the biochemical changes that may precede lung diseases, metabolomic effects on fetal and maternal lungs and plasma from rats exposed to ETS were compared to filtered air control animals. Genome- reconstructed metabolic pathways may be used to map and interpret dysregulation in metabolic networks. However, mass spectrometry-based non-targeted metabolomics datasets often comprise many metabolites for which links to enzymatic reactions have not yet been reported. Hence, network visualizations that rely on current biochemical databases are incomplete and also fail to visualize novel, structurally unidentified metabolites. Results We present a novel approach to integrate biochemical pathway and chemical relationships to map all detected metabolites in network graphs (MetaMapp) using KEGG reactant pair database, Tanimoto chemical and NIST mass spectral similarity scores. In fetal and maternal lungs, and in maternal blood plasma from pregnant rats exposed to environmental tobacco smoke (ETS), 459 unique metabolites comprising 179 structurally identified compounds were detected by gas chromatography time of flight mass spectrometry (GC-TOF MS) and BinBase data processing. MetaMapp graphs in Cytoscape showed much clearer metabolic modularity and complete content visualization compared to conventional biochemical mapping approaches. Cytoscape visualization of differential statistics results using these graphs showed that overall, fetal lung metabolism was more impaired than lungs and blood metabolism in dams. Fetuses from ETS-exposed dams expressed lower lipid and nucleotide levels and higher amounts of energy metabolism intermediates than control animals, indicating lower biosynthetic rates of metabolites for cell division, structural proteins and lipids that are critical for in lung development. Conclusions MetaMapp graphs efficiently visualizes mass spectrometry based metabolomics datasets as network graphs in Cytoscape, and highlights metabolic alterations that can be associated with higher rate of pulmonary diseases and infections in children prenatally exposed to ETS. The MetaMapp scripts can be accessed at http://metamapp.fiehnlab.ucdavis.edu. PMID:22591066

  4. Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state.

    PubMed

    Huang, Li-Shar; Shen, John T; Wang, Andy C; Berry, Edward A

    2006-01-01

    Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.

  5. Crystallographic Studies of the Binding of Ligands to theDicarboxylate Site of Complex II, and the Identity of the Ligand in the'Oxaloacetate-Inhibited' State

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

    Huang, Li-Shar; Shen, John T.; Wang, Andy C.

    2006-07-01

    Mitochondrial Complex II (succinate:ubiquinoneoxidoreductase) is purified in a partially innactivated state, which canbe activated by removal of tightly bound oxaloacetate (Kearney, E.B. etal. Biochem Biophys Res Commun 49, 1115-1121). We crystallized Complex IIin the presence of oxaloacetate or with the endogenous inhibitor bound.The structure showed a ligand essentially identical to the "malate-likeintermediate" found in Shewanella Flavocytochrome c crystallized withfumarate (Taylor, P., et al. Nat Struct Biol 6, 1108-1112.)Crystallization of Complex II in the presence of excess fumarate alsogave the malate-like intermediate or a mixture of that and fumarate atthe active site. In order to more conveniently monitor the occupationstate ofmore » the dicarboxylate site, we are developing a library of UV/Visspectral effects induced by binding different ligands to the site.Treatment with fumarate results in rapid development of the fumaratedifference spectrum and then a very slow conversion into a speciesspectrally similar to the OAA liganded complex. Complex II is known to becapable of oxidizing malate to the enol form of oxaloacetate (Belikova,Y.O., et al. Biochim Biophys Acta 936, 1-9). The observations abovesuggest it may also be capable of interconverting fumarate and malate. Itmay be useful for understanding the mechanism and regulation of theenzyme to identify the malate-like intermediate and its pathway offormation from oxaloacetate or fumarate.« less

  6. In vitro reconstitution of interactions in the CARD9 signalosome

    PubMed Central

    Park, Jin Hee; Choi, Jae Young; Mustafa, Mir Faisal; Park, Hyun Ho

    2017-01-01

    The caspase-associated recruitment domain (CARD)-containing protein 9 (CARD9) signalosome is composed of CARD9, B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). The CARD9 signalosome has been reported to exert critical functions in the immunoreceptor tyrosine-based activation motif-coupled receptor-mediated activation of myeloid cells, through nuclear factor-κB pathways during innate immunity processes. During CARD9 signalosome assembly, BCL10 has been revealed to function as an adaptor protein and to interact with CARD9 via CARD-CARD interactions; BCL10 also interacts with MALT1 via its C-terminal Ser/Thr-rich region and the first immunoglobulin domain of MALT1. The CARD9 signalosome is implicated in critical biological processes; however, its structural and biochemical characteristics have yet to be elucidated. In the present study, CARD9 and BCL10 CARDs were successfully purified and characterized, and their biochemical properties were investigated. In addition, CARD9-BCL10 complexes were reconstituted in vitro under low salt and pH conditions. Furthermore, based on structural modeling data, a scheme was proposed to describe the interactions between CARD9 and BCL10. This provides a further understanding of the mechanism of how the CARD9 signalosome may be assembled. PMID:28765954

  7. The Conserved ATPase Get3/Arr4 Modulates the Activity of Membrane-Associated Proteins in Saccharomyces cerevisiae

    PubMed Central

    Auld, Kathryn L.; Hitchcock, Amy L.; Doherty, Hugh K.; Frietze, Seth; Huang, Linda S.; Silver, Pamela A.

    2006-01-01

    The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin–proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin–proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Δget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways. PMID:16816426

  8. Role of Silicon on Plant–Pathogen Interactions

    PubMed Central

    Wang, Min; Gao, Limin; Dong, Suyue; Sun, Yuming; Shen, Qirong; Guo, Shiwei

    2017-01-01

    Although silicon (Si) is not recognized as an essential element for general higher plants, it has beneficial effects on the growth and production of a wide range of plant species. Si is known to effectively mitigate various environmental stresses and enhance plant resistance against both fungal and bacterial pathogens. In this review, the effects of Si on plant–pathogen interactions are analyzed, mainly on physical, biochemical, and molecular aspects. In most cases, the Si-induced biochemical/molecular resistance during plant–pathogen interactions were dominated as joint resistance, involving activating defense-related enzymes activates, stimulating antimicrobial compound production, regulating the complex network of signal pathways, and activating of the expression of defense-related genes. The most previous studies described an independent process, however, the whole plant resistances were rarely considered, especially the interaction of different process in higher plants. Si can act as a modulator influencing plant defense responses and interacting with key components of plant stress signaling systems leading to induced resistance. Priming of plant defense responses, alterations in phytohormone homeostasis, and networking by defense signaling components are all potential mechanisms involved in Si-triggered resistance responses. This review summarizes the roles of Si in plant–microbe interactions, evaluates the potential for improving plant resistance by modifying Si fertilizer inputs, and highlights future research concerning the role of Si in agriculture. PMID:28529517

  9. Fatty Aldehyde and Fatty Alcohol Metabolism: Review and Importance for Epidermal Structure and Function

    PubMed Central

    Rizzo, William B.

    2014-01-01

    Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren-Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. PMID:24036493

  10. Exploring the combinatorial space of complete pathways to chemicals.

    PubMed

    Wang, Lin; Ng, Chiam Yu; Dash, Satyakam; Maranas, Costas D

    2018-04-06

    Computational pathway design tools often face the challenges of balancing the stoichiometry of co-metabolites and cofactors, and dealing with reaction rule utilization in a single workflow. To this end, we provide an overview of two complementary stoichiometry-based pathway design tools optStoic and novoStoic developed in our group to tackle these challenges. optStoic is designed to determine the stoichiometry of overall conversion first which optimizes a performance criterion (e.g. high carbon/energy efficiency) and ensures a comprehensive search of co-metabolites and cofactors. The procedure then identifies the minimum number of intervening reactions to connect the source and sink metabolites. We also further the pathway design procedure by expanding the search space to include both known and hypothetical reactions, represented by reaction rules, in a new tool termed novoStoic. Reaction rules are derived based on a mixed-integer linear programming (MILP) compatible reaction operator, which allow us to explore natural promiscuous enzymes, engineer candidate enzymes that are not already promiscuous as well as design de novo enzymes. The identified biochemical reaction rules then guide novoStoic to design routes that expand the currently known biotransformation space using a single MILP modeling procedure. We demonstrate the use of the two computational tools in pathway elucidation by designing novel synthetic routes for isobutanol. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  11. A mathematical model for regulating monomer composition of the microbially synthesized polyhydroxyalkanoate copolymers.

    PubMed

    Xu, Jun; Guo, Baohua; Zhang, Zengmin; Wu, Qiong; Zhou, Quan; Chen, Jinchun; Chen, Guoqiang; Li, Guodong

    2005-06-30

    A mathematical model is proposed for predicting the copolymer composition of the microbially synthesized polyhydroxyalkanoate (PHA) copolymers. Based on the biochemical reactions involved in the precursor formation and polymerization pathways, the model correlates the copolymer composition with the cultivation conditions, the enzyme levels and selectivity, and the metabolic pathways. It suggests the following points: (1) in the case of a sole carbon source, the copolymer composition depends mainly on the topology of the metabolic pathways and the selectivity of both the enzymes involved in the precursor formation and the polymerization route; (2) the copolymer composition can be varied in a wide range via alteration of the flux ratio of different types of monomers channeled from two or more independent and simultaneous pathways; (3) the enzymes which should be over-expressed or inhibited to obtain the desired copolymer composition can be predicted. For example, inhibition of the beta-oxidation pathway will increase the content of the monomer units with longer chain length. To test the model, various experiments were envisaged by varying cultivation time, concentration and chain length of the sole carbon source, and molar ratio of the cosubstrates. The predictions from the model agree well with the experimental results. Therefore, the proposed model will be useful in predicting the PHA copolymer composition under different biochemical reaction conditions. In other words, it can provide a guide for the synthesis of desired PHA copolymers.

  12. Pathway Design, Engineering, and Optimization.

    PubMed

    Garcia-Ruiz, Eva; HamediRad, Mohammad; Zhao, Huimin

    The microbial metabolic versatility found in nature has inspired scientists to create microorganisms capable of producing value-added compounds. Many endeavors have been made to transfer and/or combine pathways, existing or even engineered enzymes with new function to tractable microorganisms to generate new metabolic routes for drug, biofuel, and specialty chemical production. However, the success of these pathways can be impeded by different complications from an inherent failure of the pathway to cell perturbations. Pursuing ways to overcome these shortcomings, a wide variety of strategies have been developed. This chapter will review the computational algorithms and experimental tools used to design efficient metabolic routes, and construct and optimize biochemical pathways to produce chemicals of high interest.

  13. Micronutrient metal speciation is driven by competitive organic chelation in grassland soils.

    NASA Astrophysics Data System (ADS)

    Boiteau, R.; Shaw, J. B.; Paša-Tolić, L.; Koppenaal, D.; Jansson, J.

    2017-12-01

    Many elements are scarcely soluble in aqueous conditions found in high pH environments, such as calcareous grassland soils, unless complexed to strong binding organic ligands. To overcome this limitation, some plants and microbes produce chelators that solubilize micronutrient metals such as Fe, Ni, Cu, and Zn from mineral phases. These complexes are taken up by organisms via specific membrane receptors, thereby differentially impacting the bioavailability of these metals to the plant and microbial community. Although the importance of these chelation strategies for individual organisms has been well established, little is known about which pathways coexist within rhizosphere microbiomes or how they interact and compete for metal binding. Identifying these metallo-organic species within natural ecosystems has remained a formidable analytical challenge due to the vast diversity of compounds and poorly defined metabolic processes in complex soil matrix. Herein, we employed recently developed liquid chromatography (LC) mass spectrometry (MS) methods to characterize the speciation of water-soluble dissolved trace elements (Fe, Ni, Cu, and Zn) from Kansas Prairie soil. Both plant and fungal chelators were identified, revealing compound-specific patterns of chelation to biologically essential metals. Numerous metabolites typically implicated in plant iron acquisition and homeostasis, including mugineic acids, deoxymugineic acid, nicotianamine, and hydroxynicotianamine, dominated the speciation of divalent metals such as Ni, Cu, and Zn (2-57 pmol / g soil). In contrast, the fungal siderophore ferricrocine bound comparatively more trivalent Fe (9pmol / g soil). These results define biochemical pathways that underpin the regulation of metals in the grassland rhizosphere. They also raise new questions about the competition of these compounds for metal binding and their bioavailability to different members of the rhizosphere population.

  14. Structured plant metabolomics for the simultaneous exploration of multiple factors.

    PubMed

    Vasilev, Nikolay; Boccard, Julien; Lang, Gerhard; Grömping, Ulrike; Fischer, Rainer; Goepfert, Simon; Rudaz, Serge; Schillberg, Stefan

    2016-11-17

    Multiple factors act simultaneously on plants to establish complex interaction networks involving nutrients, elicitors and metabolites. Metabolomics offers a better understanding of complex biological systems, but evaluating the simultaneous impact of different parameters on metabolic pathways that have many components is a challenging task. We therefore developed a novel approach that combines experimental design, untargeted metabolic profiling based on multiple chromatography systems and ionization modes, and multiblock data analysis, facilitating the systematic analysis of metabolic changes in plants caused by different factors acting at the same time. Using this method, target geraniol compounds produced in transgenic tobacco cell cultures were grouped into clusters based on their response to different factors. We hypothesized that our novel approach may provide more robust data for process optimization in plant cell cultures producing any target secondary metabolite, based on the simultaneous exploration of multiple factors rather than varying one factor each time. The suitability of our approach was verified by confirming several previously reported examples of elicitor-metabolite crosstalk. However, unravelling all factor-metabolite networks remains challenging because it requires the identification of all biochemically significant metabolites in the metabolomics dataset.

  15. Leigh Syndrome with Nephropathy and CoQ10 Deficiency Due to decaprenyl diphosphate synthase subunit 2 (PDSS2) Mutations

    PubMed Central

    López, Luis Carlos ; Schuelke, Markus ; Quinzii, Catarina M. ; Kanki, Tomotake ; Rodenburg, Richard J. T. ; Naini, Ali ; DiMauro, Salvatore ; Hirano, Michio 

    2006-01-01

    Coenzyme Q10 (CoQ10) is a vital lipophilic molecule that transfers electrons from mitochondrial respiratory chain complexes I and II to complex III. Deficiency of CoQ10 has been associated with diverse clinical phenotypes, but, in most patients, the molecular cause is unknown. The first defect in a CoQ10 biosynthetic gene, COQ2, was identified in a child with encephalomyopathy and nephrotic syndrome and in a younger sibling with only nephropathy. Here, we describe an infant with severe Leigh syndrome, nephrotic syndrome, and CoQ10 deficiency in muscle and fibroblasts and compound heterozygous mutations in the PDSS2 gene, which encodes a subunit of decaprenyl diphosphate synthase, the first enzyme of the CoQ10 biosynthetic pathway. Biochemical assays with radiolabeled substrates indicated a severe defect in decaprenyl diphosphate synthase in the patient’s fibroblasts. This is the first description of pathogenic mutations in PDSS2 and confirms the molecular and clinical heterogeneity of primary CoQ10 deficiency. PMID:17186472

  16. Hepatic Steatosis as a Marker of Metabolic Dysfunction

    PubMed Central

    Fabbrini, Elisa; Magkos, Faidon

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is the liver manifestation of the complex metabolic derangements associated with obesity. NAFLD is characterized by excessive deposition of fat in the liver (steatosis) and develops when hepatic fatty acid availability from plasma and de novo synthesis exceeds hepatic fatty acid disposal by oxidation and triglyceride export. Hepatic steatosis is therefore the biochemical result of an imbalance between complex pathways of lipid metabolism, and is associated with an array of adverse changes in glucose, fatty acid, and lipoprotein metabolism across all tissues of the body. Intrahepatic triglyceride (IHTG) content is therefore a very good marker (and in some cases may be the cause) of the presence and the degree of multiple-organ metabolic dysfunction. These metabolic abnormalities are likely responsible for many cardiometabolic risk factors associated with NAFLD, such as insulin resistance, type 2 diabetes mellitus, and dyslipidemia. Understanding the factors involved in the pathogenesis and pathophysiology of NAFLD will lead to a better understanding of the mechanisms responsible for the metabolic complications of obesity, and hopefully to the discovery of novel effective treatments for their reversal. PMID:26102213

  17. Differential Attenuation of NMR Signals by Complementary Ion-Exchange Resin Beads for De Novo Analysis of Complex Metabolomics Mixtures.

    PubMed

    Zhang, Bo; Yuan, Jiaqi; Brüschweiler, Rafael

    2017-07-12

    A primary goal of metabolomics is the characterization of a potentially very large number of metabolites that are part of complex mixtures. Application to biofluids and tissue samples offers insights into biochemical metabolic pathways and their role in health and disease. 1D 1 H and 2D 13 C- 1 H HSQC NMR spectra are most commonly used for this purpose. They yield quantitative information about each proton of the mixture, but do not tell which protons belong to the same molecule. Interpretation requires the use of NMR spectral databases, which naturally limits these investigations to known metabolites. Here, a new method is presented that uses complementary ion exchange resin beads to differentially attenuate 2D NMR cross-peaks that belong to different metabolites. Based on their characteristic attenuation patterns, cross-peaks could be clustered and assigned to individual molecules, including unknown metabolites with multiple spin systems, as demonstrated for a metabolite model mixture and E. coli cell lysate. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning

    PubMed Central

    Manatschal, Cristina; Farcas, Ana-Maria; Degen, Miriam Steiner; Bayer, Mathias; Kumar, Anil; Landgraf, Christiane; Volkmer, Rudolf; Barral, Yves; Steinmetz, Michel O.

    2016-01-01

    The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue). Site1 and Site2 contain SxIP motifs; however, Site3 defines a novel type of EB1-binding site. Whereas Site2 and Site3 mediate Kar9 recruitment to microtubule tips, nuclear movement, and karyogamy, only Site2 functions in spindle positioning during metaphase. Site1 in turn plays an inhibitory role during mating. Additionally, the Kar9-Bim1 complex is involved in microtubule-independent activities during mating. Together, our data reveal how multiple and partially redundant EB1-binding sites provide a microtubule-associated protein with the means to modulate its biochemical properties to promote different molecular processes during cell proliferation and differentiation. PMID:27682587

  19. Single-molecule studies of multi-protein machines

    NASA Astrophysics Data System (ADS)

    van Oijen, Antoine

    2010-03-01

    Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. Our group is developing the single-molecule tools that will make it possible to study biochemical pathways of arbitrary complexity at the single-molecule level. I will discuss results of single-molecule experiments on the replisome, the molecular machinery that is responsible for replication of DNA. We stretch individual DNA molecules and use their elastic properties to obtain dynamic information on the proteins that unwind the double helix and copy its genetic information. Furthermore, we visualize fluorescently labeled components of the replisome and thus obtain information on stochiometry and exchange kinetics. This simultaneous observation of catalytic activity and composition allows us to gain deeper insight into the structure-function relationship of the replisome.

  20. Trio’s Rho-specific GEF domain is the missing Gαq effector in C. elegans

    PubMed Central

    Williams, Stacey L.; Lutz, Susanne; Charlie, Nicole K.; Vettel, Christiane; Ailion, Michael; Coco, Cassandra; Tesmer, John J.G.; Jorgensen, Erik M.; Wieland, Thomas; Miller, Kenneth G.

    2007-01-01

    The Gαq pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cβ [PLCβ]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Gαq pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCβ). Strains containing null mutations in both EGL-8 (PLCβ) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Gαq-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Gαq synergizes with Trio RhoGEF to activate RhoA. Activated Gαq and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio’s Rho-specific GEF domain is a major Gαq effector that, together with PLCβ, mediates the Gαq signaling that drives the locomotion, egg laying, and growth of the animal. PMID:17942708

  1. The complex spine: the multidimensional system of causal pathways for low-back disorders.

    PubMed

    Marras, William S

    2012-12-01

    The aim of this study was to examine the logic behind the knowledge of low-back problem causal pathways. Low-back pain and low-back disorders (LBDs) continue to represent the major musculoskeletal risk problem in the workplace,with the prevalence and costs of such disorders increasing over time. In recent years, there has been much criticism of the ability of ergonomics methods to control the risk of LBDs. Logical assessment of the systems logic associated with our understanding and prevention of LBDs. Current spine loading as well as spine tolerance research efforts are bringing the field to the point where there is a better systems understanding of the inextricable link between the musculoskeletal system and the cognitive system. Loading is influenced by both the physical environment factors as well as mental demands, whereas tolerances are defined by both physical tissue tolerance and biochemically based tissue sensitivities to pain. However, the logic used in many low-back risk assessment tools may be overly simplistic, given what is understood about causal pathways. Current tools typically assess only load or position in a very cursory manner. Efforts must work toward satisfying both the physical environment and the cognitive environment for the worker if one is to reliably lower the risk of low-back problems. This systems representation of LBD development may serve as a guide to identify gaps in our understanding of LBDs.

  2. The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

    PubMed Central

    Walley, Justin W.; Rowe, Heather C.; Xiao, Yanmei; Chehab, E. Wassim; Kliebenstein, Daniel J.; Wagner, Doris; Dehesh, Katayoon

    2008-01-01

    Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks. PMID:19079584

  3. Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-07-05

    The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.

  4. Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

    PubMed Central

    Yoshida, Keisuke; Hisabori, Toru

    2016-01-01

    The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

  5. The chromatin remodeler SPLAYED regulates specific stress signaling pathways.

    PubMed

    Walley, Justin W; Rowe, Heather C; Xiao, Yanmei; Chehab, E Wassim; Kliebenstein, Daniel J; Wagner, Doris; Dehesh, Katayoon

    2008-12-01

    Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.

  6. In Situ Tracking Kinetic Pathways of Li + /Na + Substitution during Ion-Exchange Synthesis of Li xNa 1.5–x VOPO 4 F 0.5

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

    Park, Young-Uk; Bai, Jianming; Wang, Liping

    Ion exchange is a ubiquitous phenomenon central to wide industrial applications, ranging from traditional (bio)chemical separation to the emerging chimie douce synthesis of materials for batteries and other energy applications. The exchange process is complex, involving substitution and transport of different ions under non-equilibrium conditions, and thus difficult to probe, leaving a gap in mechanistic understanding of kinetic exchange pathways toward final products. Herein, we report in situ tracking kinetic pathways of Li +/Na + substitution during solvothermal ion-exchange synthesis of Li xNa 1.5-xVOPO 4F 0.5 (0 ≤ x ≤ 1.5), a promising multi-Li polyanionic cathode for batteries. The real-timemore » observation, corroborated by first-principles calculations, reveals a selective replacement of Na + by Li +, leading to peculiar Na +/Li +/vacancy orderings in the intermediates. Contradicting the traditional belief of facile topotactic substitution via solid solution reaction, an abrupt two-phase transformation occurs and predominantly governs the kinetics of ion exchange and transport in the 1D polyanionic framework, consequently leading to significant difference of Li stoichiometry and electrochemical properties in the exchanged products. The findings may help to pave the way for rational design of ion exchange synthesis for making new materials.« less

  7. Transport of phosphatidylserine from the endoplasmic reticulum to the site of phosphatidylserine decarboxylase2 in yeast.

    PubMed

    Kannan, Muthukumar; Riekhof, Wayne R; Voelker, Dennis R

    2015-02-01

    Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. In Situ Tracking Kinetic Pathways of Li + /Na + Substitution during Ion-Exchange Synthesis of Li xNa 1.5–x VOPO 4 F 0.5

    DOE PAGES

    Park, Young-Uk; Bai, Jianming; Wang, Liping; ...

    2017-08-29

    Ion exchange is a ubiquitous phenomenon central to wide industrial applications, ranging from traditional (bio)chemical separation to the emerging chimie douce synthesis of materials for batteries and other energy applications. The exchange process is complex, involving substitution and transport of different ions under non-equilibrium conditions, and thus difficult to probe, leaving a gap in mechanistic understanding of kinetic exchange pathways toward final products. Herein, we report in situ tracking kinetic pathways of Li +/Na + substitution during solvothermal ion-exchange synthesis of Li xNa 1.5-xVOPO 4F 0.5 (0 ≤ x ≤ 1.5), a promising multi-Li polyanionic cathode for batteries. The real-timemore » observation, corroborated by first-principles calculations, reveals a selective replacement of Na + by Li +, leading to peculiar Na +/Li +/vacancy orderings in the intermediates. Contradicting the traditional belief of facile topotactic substitution via solid solution reaction, an abrupt two-phase transformation occurs and predominantly governs the kinetics of ion exchange and transport in the 1D polyanionic framework, consequently leading to significant difference of Li stoichiometry and electrochemical properties in the exchanged products. The findings may help to pave the way for rational design of ion exchange synthesis for making new materials.« less

  9. Expansion, retention and loss in the Acyl-CoA synthetase "Bubblegum" (Acsbg) gene family in vertebrate history.

    PubMed

    Lopes-Marques, Mónica; Machado, André M; Ruivo, Raquel; Fonseca, Elza; Carvalho, Estela; Castro, L Filipe C

    2018-07-20

    Fatty acids (FAs) constitute a considerable fraction of all lipid molecules with a fundamental role in numerous physiological processes. In animals, the majority of complex lipid molecules are derived from the transformation of FAs through several biochemical pathways. Yet, for FAs to enroll in these pathways they require an activation step. FA activation is catalyzed by the rate limiting action of Acyl-CoA synthases. Several Acyl-CoA enzyme families have been previously described and classified according to the chain length of FAs they process. Here, we address the evolutionary history of the ACSBG gene family which activates, FAs with >16 carbons. Currently, two different ACSBG gene families, ACSBG1 and ACSBG2, are recognized in vertebrates. We provide evidence that a wider and unequal ACSBG gene repertoire is present in vertebrate lineages. We identify a novel ACSBG-like gene lineage which occurs specifically in amphibians, ray finned fishes, coelacanths and cartilaginous fishes named ACSBG3. Also, we show that the ACSBG2 gene lineage duplicated in the Theria ancestor. Our findings, thus offer a far richer understanding on FA activation in vertebrates and provide key insights into the relevance of comparative and functional analysis to perceive physiological differences, namely those related with lipid metabolic pathways. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Computational Insight Into the Structural Organization of Full-Length Toll-Like Receptor 4 Dimer in a Model Phospholipid Bilayer

    PubMed Central

    Patra, Mahesh Chandra; Kwon, Hyuk-Kwon; Batool, Maria; Choi, Sangdun

    2018-01-01

    Toll-like receptors (TLRs) are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM), and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4—a widely studied member of the interleukin-1 receptor/TLR superfamily—using homology modeling, protein–protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway. PMID:29593733

  11. Multimodal drugs and their future for Alzheimer's and Parkinson's disease.

    PubMed

    Van der Schyf, Cornelis J; Geldenhuys, Werner J

    2011-01-01

    This chapter discusses the rationale for developing multimodal or multifunctional drugs (also called designed multiple ligands or DMLs) aimed at disease-modifying treatment strategies for the most common neurodegenerative diseases Alzheimer's and Parkinson's disease (AD and PD). Both the prevalence and incidence of AD and PD have seen consistent and dramatic increases, a disconcerting phenomenon which, ironically, has been attributed to extended life expectancy brought about by better health care globally. In spite of these statistics, the development and introduction to the clinic of new therapies proven to prevent or delay the onset of AD and PD have been disappointing. Evidence has accumulated to suggest that the etiopathology of these diseases is extremely complex, with an array of potential drug targets located within a number of deleterious biochemical pathways. Therefore, in these diseases, it is unlikely that the complex pathoetiological cascade leading to disease initiation or progression will be mitigated by any one drug acting on a single pathway or target. The pursuit of novel DMLs may offer far better outcomes. Although certainly not the only, and perhaps not even the best, approach but farthest along the drug development pipeline in the DML paradigm are drugs that combine inhibition of monoamine oxidase with associated etiological targets unique to either AD or PD. These compounds will constitute the major focus of this chapter, which will also explore radically new paradigms that seek to combine cognitive enhancers with proneurogenesis compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

    PubMed Central

    Dhawan, Neil S.; scopton, Alex P.; Dar, Arvin C.

    2016-01-01

    Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies1. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes2. Kinase suppressor of Ras (KSR) is a MAPK scaffold3–5 that is subject to allosteric regulation through dimerization with RAF6,7. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers. PMID:27556948

  13. The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis.

    PubMed

    Qin, Gaihua; Xu, Chunyan; Ming, Ray; Tang, Haibao; Guyot, Romain; Kramer, Elena M; Hu, Yudong; Yi, Xingkai; Qi, Yongjie; Xu, Xiangyang; Gao, Zhenghui; Pan, Haifa; Jian, Jianbo; Tian, Yinping; Yue, Zhen; Xu, Yiliu

    2017-09-01

    Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. We have sequenced and assembled the pomegranate genome with 328 Mb anchored into nine pseudo-chromosomes and annotated 29 229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  14. Thermodynamic considerations on Ca2+-induced biochemical reactions in living cells

    NASA Astrophysics Data System (ADS)

    Lucia, Umberto; Ponzetto, Antonio

    2016-02-01

    Cells can be regarded as complex engines that execute a series of chemical reactions. Energy transformations, thermo-electro-chemical processes and transport phenomena can occur across cell membranes. Different, related thermo-electro-biochemical behaviour can occur between health and disease states. Analysis of the irreversibility related to ion fluxes can represent a new approach to study and control the biochemical behaviour of living cells.

  15. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

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

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases thatmore » includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in their active sites that help explain the variations in their respective substrate specificities.« less

  16. Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway.

    PubMed

    Boyd, Eric S; Thomas, Khaleh M; Dai, Yuyuan; Boyd, Jeff M; Outten, F Wayne

    2014-09-23

    Iron-sulfur (Fe-S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe-S clusters and the fundamental requirement for Fe-S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth's atmosphere. Intriguingly, Fe-S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe-S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe-S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe-S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB-SufC scaffold complex. This analysis provides a new framework for the study of Fe-S cluster biogenesis pathways and Fe-S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen.

  17. Henry Friesen Award Lecture. Work, the clinician-scientist and human biochemical genetics.

    PubMed

    Scriver, C R

    2001-08-01

    The pursuit of human biochemical genetics has allowed us to understand better how the person with the (genetic) disease differs from the disease the person has and to develop the concept that genetics belongs in all aspects of health care. It is a perspective that comes quite readily to the clinician-scientist, and the restoration of that "species" in the era of functional genomics is strongly recommended. Garrod, the initial founder of human "biochemical genetics" belonged to the clinician-scientist community. Archibald Edward Garrod introduced a paradigm, new for its day, in medicine: biochemistry is dynamic and different from the static nature of organic chemistry. It led him to think about metabolic pathways and to recognize that variation in Mendelian heredity could explain an "inborn error of metabolism." At the time, Garrod had no idea about the nature of a gene. Genes are now well understood; genomes are being described for one organism after another (including Homo sapiens) and it is understood that genomes "speak biochemistry (not phenotype)." Accordingly, in the era of genomics, biochemistry and physiology become the bases of functional genomics, and it is possible to appreciate why "nothing in biology makes sense without evolution" (and nothing in medicine will make sense without biology). Mendelian, biochemical and molecular genetics together have revealed what lies behind the 4 canonical inborn errors described by Garrod (albinisn, alkaptonuria, cystinuria and pentosuria). Both older and newer ideas in genetics, new tools for applying them (and renewed respect for the clinician-scientist) will enhance our understanding of the human biological variation that accounts for variant states of health and overt disease. A so-called monogenic phenotype (phenylketonuria) is used to illustrate, in some detail, that all disease phenotypes are, in one way or another, likely to be complex in nature. What can be known and what ought to be done, with knowledge about human genetics, to benefit individuals, families and communities (society), is both opportunity and challenge.

  18. Preliminary study on Emodin alleviating alpha-naphthylisothiocyanate-induced intrahepatic cholestasis by regulation of liver farnesoid X receptor pathway.

    PubMed

    Ding, Yan; Xiong, Xiao-Li; Zhou, Li-Shan; Yan, Su-Qi; Qin, Huan; Li, Hua-Rong; Zhang, Ling-Ling; Chen, Peng; Yao, Cong; Jiang, Zhi-Xia; Zhao, Lei

    2016-12-01

    The aim of this study is to investigate Emodin on alleviating intrahepatic cholestasis by regulation of liver farnesoid X receptor (FXR) pathway. Cell and animal models of intrahepatic cholestatis were established. Biochemical tests and histomorphology were performed. The messenger RNA (mRNA) and protein expression of FXR, small heterodimer partner (SHP), uridine diphosphate glucuronosyltransferase 2 family polypeptide B4 (UGT2B4), and bile salt export pump (BSEP) was detected. As a result, compared with the model group, the serum levels of biochemical test were significantly lower in the Emodin group (P <0.01). The histopathological changes were remitted significantly by Emodin treatment. In the model group, the mRNA and protein expression of FXR, SHP, UGT2B4, and BSEP was significantly lower than in the normal group in cell models (P <0.05). With Emodin intervention, the expression of FXR, SHP, UGT2B4, and BSEP was notably increased (P <0.05). In conclusion, Emodin plays a protective role in intrahepatic cholestasis by promoting FXR signal pathways. © The Author(s) 2016.

  19. Integrated metabolomics and proteomics highlight altered nicotinamide and polyamine pathways in lung adenocarcinoma

    PubMed Central

    Fahrmann, Johannes F.; Grapov, Dmitry; Wanichthanarak, Kwanjeera; DeFelice, Brian C.; Salemi, Michelle R.; Rom, William N.; Gandara, David R.; Phinney, Brett S.; Fiehn, Oliver; Pass, Harvey

    2017-01-01

    Abstract Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma (1): increased glycosylation and glutaminolysis (2); elevated Nrf2 activation (3); increase in nicotinic and nicotinamide salvaging pathways and (4) elevated polyamine biosynthesis linked to differential regulation of the s-adenosylmethionine/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompany early stage lung tumorigenesis and highlight potential therapeutic targets. PMID:28049629

  20. Identification of the First Riboflavin Catabolic Gene Cluster Isolated from Microbacterium maritypicum G10*

    PubMed Central

    Xu, Hui; Chakrabarty, Yindrila; Philmus, Benjamin; Mehta, Angad P.; Bhandari, Dhananjay; Hohmann, Hans-Peter; Begley, Tadhg P.

    2016-01-01

    Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed. PMID:27590337

  1. Targeting the Hippo signalling pathway for cancer treatment.

    PubMed

    Nakatani, Keisuke; Maehama, Tomohiko; Nishio, Miki; Goto, Hiroki; Kato, Wakako; Omori, Hirofumi; Miyachi, Yosuke; Togashi, Hideru; Shimono, Yohei; Suzuki, Akira

    2017-03-01

    The Hippo signalling pathway monitors cell-cell contact and external factors that shape tissue structure. In mice, tumourigenesis and developmental abnormalities are common consequences of dysregulated Hippo signalling. Expression of Hippo pathway components is also frequently altered in human tumours and correlates with poor prognosis and reduced patient survival. Thus, the Hippo pathway is an attractive anti-cancer target. Here, we provide an overview of the function and regulation of Hippo signalling components and summarize progress to date on the development of agents able to regulate Hippo signalling for cancer therapy. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  2. In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv. Carlos Reveals Proteins Associated with Flavor and Aroma Compounds.

    PubMed

    Kambiranda, Devaiah; Basha, Sheikh M; Singh, Rakesh K; He, Huan; Calvin, Kate; Mercer, Roger

    2016-09-02

    Ripening in nonclimacteric fruits such as grape involves complex chemical changes that have a profound influence on the accumulation of flavor and aroma compounds distinct to a particular grape genotype. In this study, proteome characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos), primarily grown in the Southeastern United States was performed during berry ripening. Stage-specific protein expression was obtained among different stages of berries. Differential analysis showed the expression of 522 proteins that regulate diverse biological processes and metabolic pathways. Of these, 30 proteins are associated with the production of key phenolic compounds, whereas 25 are associated with the production of muscadine aroma compounds. These proteins are involved in the phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin, and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of muscadine grape to improve its flavor and aroma properties.

  3. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

    PubMed

    Mast, Alan E

    2016-01-01

    Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients. © 2015 American Heart Association, Inc.

  4. Gsk3 Signalling and Redox Status in Bipolar Disorder: Evidence from Lithium Efficacy

    PubMed Central

    2016-01-01

    Objective. To discuss the link between glycogen synthase kinase-3 (GSK3) and the main biological alterations demonstrated in bipolar disorder (BD), with special attention to the redox status and the evidence supporting the efficacy of lithium (a GSK3 inhibitor) in the treatment of BD. Methods. A literature research on the discussed topics, using Pubmed and Google Scholar, has been conducted. Moreover, a manual selection of interesting references from the identified articles has been performed. Results. The main biological alterations of BD, pertaining to inflammation, oxidative stress, membrane ion channels, and circadian system, seem to be intertwined. The dysfunction of the GSK3 signalling pathway is involved in all the aforementioned “biological causes” of BD. In a complex scenario, it can be seen as the common denominator linking them all. Lithium inhibition of GSK3 could, at least in part, explain its positive effect on these biological dysfunctions and its superiority in terms of clinical efficacy. Conclusions. Deepening the knowledge on the molecular bases of BD is fundamental to identifying the biochemical pathways that must be targeted in order to provide patients with increasingly effective therapeutic tools against an invalidating disorder such as BD. PMID:27630757

  5. Gsk3 Signalling and Redox Status in Bipolar Disorder: Evidence from Lithium Efficacy.

    PubMed

    Luca, Antonina; Calandra, Carmela; Luca, Maria

    2016-01-01

    Objective. To discuss the link between glycogen synthase kinase-3 (GSK3) and the main biological alterations demonstrated in bipolar disorder (BD), with special attention to the redox status and the evidence supporting the efficacy of lithium (a GSK3 inhibitor) in the treatment of BD. Methods. A literature research on the discussed topics, using Pubmed and Google Scholar, has been conducted. Moreover, a manual selection of interesting references from the identified articles has been performed. Results. The main biological alterations of BD, pertaining to inflammation, oxidative stress, membrane ion channels, and circadian system, seem to be intertwined. The dysfunction of the GSK3 signalling pathway is involved in all the aforementioned "biological causes" of BD. In a complex scenario, it can be seen as the common denominator linking them all. Lithium inhibition of GSK3 could, at least in part, explain its positive effect on these biological dysfunctions and its superiority in terms of clinical efficacy. Conclusions. Deepening the knowledge on the molecular bases of BD is fundamental to identifying the biochemical pathways that must be targeted in order to provide patients with increasingly effective therapeutic tools against an invalidating disorder such as BD.

  6. A novel small molecule methyltransferase is important for virulence in Candida albicans.

    PubMed

    Lissina, Elena; Weiss, David; Young, Brian; Rella, Antonella; Cheung-Ong, Kahlin; Del Poeta, Maurizio; Clarke, Steven G; Giaever, Guri; Nislow, Corey

    2013-12-20

    Candida albicans is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of C. albicans pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in C. albicans are small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates, and regulating intra- and interspecies interactions. In this study, we demonstrated that C. albicans Crg1 (CaCrg1) is a bona fide smMTase that interacts with the toxin in vitro and in vivo. We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation, and membrane trafficking. Biochemical and genetic analyses showed that CaCrg1 plays a role in the complex sphingolipid pathway: it binds to exogenous short-chain ceramides in vitro and interacts genetically with genes of glucosylceramide pathway, and the deletion of CaCRG1 leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth Galleria mellonella, a model of infection.

  7. Herbicide-related signaling in plants reveals novel insights for herbicide use strategies, environmental risk assessment and global change assessment challenges.

    PubMed

    Alberto, Diana; Serra, Anne-Antonella; Sulmon, Cécile; Gouesbet, Gwenola; Couée, Ivan

    2016-11-01

    Herbicide impact is usually assessed as the result of a unilinear mode of action on a specific biochemical target with a typical dose-response dynamics. Recent developments in plant molecular signaling and crosstalk between nutritional, hormonal and environmental stress cues are however revealing a more complex picture of inclusive toxicity. Herbicides induce large-scale metabolic and gene-expression effects that go far beyond the expected consequences of unilinear herbicide-target-damage mechanisms. Moreover, groundbreaking studies have revealed that herbicide action and responses strongly interact with hormone signaling pathways, with numerous regulatory protein-kinases and -phosphatases, with metabolic and circadian clock regulators and with oxidative stress signaling pathways. These interactions are likely to result in mechanisms of adjustment that can determine the level of sensitivity or tolerance to a given herbicide or to a mixture of herbicides depending on the environmental and developmental status of the plant. Such regulations can be described as rheostatic and their importance is discussed in relation with herbicide use strategies, environmental risk assessment and global change assessment challenges. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

    NASA Astrophysics Data System (ADS)

    Kaushik, Aman C.; Kumar, Sanjay; Wei, Dong Q.; Sahi, Shakti

    2018-02-01

    GPR142 (G protein receptor 142) is a novel orphan GPCR (G protein coupled receptor) belonging to ‘Class A’ of GPCR family and expressed in beta cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

  9. SENESCENCE (AGEING) @ 2011

    PubMed Central

    Nigam, Anjana

    2011-01-01

    Ageing, also called as senescence, is one of the most complex, intrinsic, biological processes of growing older and resulting into reduced functional ability of the organism. Telomerase, environment, low calorie diets, free radicals, etc., are all believed to affect this ageing process. A number of genetic components of ageing have been identified using model organisms. Genes, mainly the sirtuins, regulate the ageing speed by indirection and controlling organism resistance to damages by exogenous and endogenous stresses. In higher organisms, ageing is likely to be regulated, in part, through the insulin/insulin-like growth factor 1 pathway. Besides this, the induction of apoptosis in stem and progenitor cells, increased p53 activity, and autophagy is also thought to trigger premature organismal ageing. Ageing has also been shown to upregulate expression of inflammatory mediators in mouse adipose tissue. The understanding of pathophysiology of ageing over the past few years has posed tremendous challenges for the development of anti-ageing medicine for targeted therapy. Future research areas must include targeted role of systemic inflammatory markers such as C-reactive protein and interleukin 6 and other biochemical and genetic studies including gene signaling pathways, gene microarray analysis, gene modulation, gene therapy, and development of animal/human models for potential therapeutic measures and evaluations. PMID:22345758

  10. Lyapunov exponents and phase diagrams reveal multi-factorial control over TRAIL-induced apoptosis

    PubMed Central

    Aldridge, Bree B; Gaudet, Suzanne; Lauffenburger, Douglas A; Sorger, Peter K

    2011-01-01

    Receptor-mediated apoptosis proceeds via two pathways: one requiring only a cascade of initiator and effector caspases (type I behavior) and the second requiring an initiator–effector caspase cascade and mitochondrial outer membrane permeabilization (type II behavior). Here, we investigate factors controlling type I versus II phenotypes by performing Lyapunov exponent analysis of an ODE-based model of cell death. The resulting phase diagrams predict that the ratio of XIAP to pro-caspase-3 concentrations plays a key regulatory role: type I behavior predominates when the ratio is low and type II behavior when the ratio is high. Cell-to-cell variability in phenotype is observed when the ratio is close to the type I versus II boundary. By positioning multiple tumor cell lines on the phase diagram we confirm these predictions. We also extend phase space analysis to mutations affecting the rate of caspase-3 ubiquitylation by XIAP, predicting and showing that such mutations abolish all-or-none control over activation of effector caspases. Thus, phase diagrams derived from Lyapunov exponent analysis represent a means to study multi-factorial control over a complex biochemical pathway. PMID:22108795

  11. A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil1[C][W][OA

    PubMed Central

    Berim, Anna; Hyatt, David C.; Gang, David R.

    2012-01-01

    Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 4′- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4′-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation. PMID:22923679

  12. A Comparison of Blood Factor XII Autoactivation in Buffer, Protein Cocktail, Serum, and Plasma Solutions

    PubMed Central

    Golas, Avantika; Yeh, Chyi-Huey Josh; Pitakjakpipop, Harit; Siedlecki, Christopher A.; Vogler, Erwin A.

    2012-01-01

    Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212

  13. Comparative life cycle assessment of lignocellulosic ethanol production: biochemical versus thermochemical conversion.

    PubMed

    Mu, Dongyan; Seager, Thomas; Rao, P Suresh; Zhao, Fu

    2010-10-01

    Lignocellulosic biomass can be converted into ethanol through either biochemical or thermochemical conversion processes. Biochemical conversion involves hydrolysis and fermentation while thermochemical conversion involves gasification and catalytic synthesis. Even though these routes produce comparable amounts of ethanol and have similar energy efficiency at the plant level, little is known about their relative environmental performance from a life cycle perspective. Especially, the indirect impacts, i.e. emissions and resource consumption associated with the production of various process inputs, are largely neglected in previous studies. This article compiles material and energy flow data from process simulation models to develop life cycle inventory and compares the fossil fuel consumption, greenhouse gas emissions, and water consumption of both biomass-to-ethanol production processes. The results are presented in terms of contributions from feedstock, direct, indirect, and co-product credits for four representative biomass feedstocks i.e., wood chips, corn stover, waste paper, and wheat straw. To explore the potentials of the two conversion pathways, different technological scenarios are modeled, including current, 2012 and 2020 technology targets, as well as different production/co-production configurations. The modeling results suggest that biochemical conversion has slightly better performance on greenhouse gas emission and fossil fuel consumption, but that thermochemical conversion has significantly less direct, indirect, and life cycle water consumption. Also, if the thermochemical plant operates as a biorefinery with mixed alcohol co-products separated for chemicals, it has the potential to achieve better performance than biochemical pathway across all environmental impact categories considered due to higher co-product credits associated with chemicals being displaced. The results from this work serve as a starting point for developing full life cycle assessment model that facilitates effective decision-making regarding lignocellulosic ethanol production.

  14. Comparative Life Cycle Assessment of Lignocellulosic Ethanol Production: Biochemical Versus Thermochemical Conversion

    NASA Astrophysics Data System (ADS)

    Mu, Dongyan; Seager, Thomas; Rao, P. Suresh; Zhao, Fu

    2010-10-01

    Lignocellulosic biomass can be converted into ethanol through either biochemical or thermochemical conversion processes. Biochemical conversion involves hydrolysis and fermentation while thermochemical conversion involves gasification and catalytic synthesis. Even though these routes produce comparable amounts of ethanol and have similar energy efficiency at the plant level, little is known about their relative environmental performance from a life cycle perspective. Especially, the indirect impacts, i.e. emissions and resource consumption associated with the production of various process inputs, are largely neglected in previous studies. This article compiles material and energy flow data from process simulation models to develop life cycle inventory and compares the fossil fuel consumption, greenhouse gas emissions, and water consumption of both biomass-to-ethanol production processes. The results are presented in terms of contributions from feedstock, direct, indirect, and co-product credits for four representative biomass feedstocks i.e., wood chips, corn stover, waste paper, and wheat straw. To explore the potentials of the two conversion pathways, different technological scenarios are modeled, including current, 2012 and 2020 technology targets, as well as different production/co-production configurations. The modeling results suggest that biochemical conversion has slightly better performance on greenhouse gas emission and fossil fuel consumption, but that thermochemical conversion has significantly less direct, indirect, and life cycle water consumption. Also, if the thermochemical plant operates as a biorefinery with mixed alcohol co-products separated for chemicals, it has the potential to achieve better performance than biochemical pathway across all environmental impact categories considered due to higher co-product credits associated with chemicals being displaced. The results from this work serve as a starting point for developing full life cycle assessment model that facilitates effective decision-making regarding lignocellulosic ethanol production.

  15. A Synthetic Alternative to Canonical One-Carbon Metabolism.

    PubMed

    Bouzon, Madeleine; Perret, Alain; Loreau, Olivier; Delmas, Valérie; Perchat, Nadia; Weissenbach, Jean; Taran, Frédéric; Marlière, Philippe

    2017-08-18

    One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO 2 . The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

  16. The Molecular Ecophysiology of Programmed Cell Death in Marine Phytoplankton

    NASA Astrophysics Data System (ADS)

    Bidle, Kay D.

    2015-01-01

    Planktonic, prokaryotic, and eukaryotic photoautotrophs (phytoplankton) share a diverse and ancient evolutionary history, during which time they have played key roles in regulating marine food webs, biogeochemical cycles, and Earth's climate. Because phytoplankton represent the basis of marine ecosystems, the manner in which they die critically determines the flow and fate of photosynthetically fixed organic matter (and associated elements), ultimately constraining upper-ocean biogeochemistry. Programmed cell death (PCD) and associated pathway genes, which are triggered by a variety of nutrient stressors and are employed by parasitic viruses, play an integral role in determining the cell fate of diverse photoautotrophs in the modern ocean. Indeed, these multifaceted death pathways continue to shape the success and evolutionary trajectory of diverse phytoplankton lineages at sea. Research over the past two decades has employed physiological, biochemical, and genetic techniques to provide a novel, comprehensive, mechanistic understanding of the factors controlling this key process. Here, I discuss the current understanding of the genetics, activation, and regulation of PCD pathways in marine model systems; how PCD evolved in unicellular photoautotrophs; how it mechanistically interfaces with viral infection pathways; how stress signals are sensed and transduced into cellular responses; and how novel molecular and biochemical tools are revealing the impact of PCD genes on the fate of natural phytoplankton assemblages.

  17. Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

    PubMed Central

    Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

    2013-01-01

    Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

  18. An Inductive Logic Programming Approach to Validate Hexose Binding Biochemical Knowledge.

    PubMed

    Nassif, Houssam; Al-Ali, Hassan; Khuri, Sawsan; Keirouz, Walid; Page, David

    2010-01-01

    Hexoses are simple sugars that play a key role in many cellular pathways, and in the regulation of development and disease mechanisms. Current protein-sugar computational models are based, at least partially, on prior biochemical findings and knowledge. They incorporate different parts of these findings in predictive black-box models. We investigate the empirical support for biochemical findings by comparing Inductive Logic Programming (ILP) induced rules to actual biochemical results. We mine the Protein Data Bank for a representative data set of hexose binding sites, non-hexose binding sites and surface grooves. We build an ILP model of hexose-binding sites and evaluate our results against several baseline machine learning classifiers. Our method achieves an accuracy similar to that of other black-box classifiers while providing insight into the discriminating process. In addition, it confirms wet-lab findings and reveals a previously unreported Trp-Glu amino acids dependency.

  19. Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.

    PubMed

    Schild, Florie; Kieffer-Jaquinod, Sylvie; Palencia, Andrés; Cobessi, David; Sarret, Géraldine; Zubieta, Chloé; Jourdain, Agnès; Dumas, Renaud; Forge, Vincent; Testemale, Denis; Bourguignon, Jacques; Hugouvieux, Véronique

    2014-11-14

    The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Biochemical and Biophysical Characterization of the Selenium-binding and Reducing Site in Arabidopsis thaliana Homologue to Mammals Selenium-binding Protein 1*

    PubMed Central

    Schild, Florie; Kieffer-Jaquinod, Sylvie; Palencia, Andrés; Cobessi, David; Sarret, Géraldine; Zubieta, Chloé; Jourdain, Agnès; Dumas, Renaud; Forge, Vincent; Testemale, Denis; Bourguignon, Jacques; Hugouvieux, Véronique

    2014-01-01

    The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO32−) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys21 and Cys22 as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO32− to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism. PMID:25274629

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