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

Comparative genomics approaches to understanding and manipulating plant metabolism.

PubMed

Bradbury, Louis M T; Niehaus, Tom D; Hanson, Andrew D

2013-04-01

Over 3000 genomes, including numerous plant genomes, are now sequenced. However, their annotation remains problematic as illustrated by the many conserved genes with no assigned function, vague annotations such as 'kinase', or even wrong ones. Around 40% of genes of unknown function that are conserved between plants and microbes are probably metabolic enzymes or transporters; finding functions for these genes is a major challenge. Comparative genomics has correctly predicted functions for many such genes by analyzing genomic context, and gene fusions, distributions and co-expression. Comparative genomics complements genetic and biochemical approaches to dissect metabolism, continues to increase in power and decrease in cost, and has a pivotal role in modeling and engineering by helping identify functions for all metabolic genes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Proliferating cell nuclear antigen in the cytoplasm interacts with components of glycolysis and cancer.

PubMed

Naryzhny, Stanislav N; Lee, Hoyun

2010-10-22

Proliferating cell nuclear antigen (PCNA) is involved in a wide range of functions in the nucleus. However, a substantial amount of PCNA is also present in the cytoplasm, although their function is unknown. Here we show, through Far-Western blotting and mass spectrometry, that PCNA is associated with several cytoplasmic oncoproteins, including elongation factor, malate dehydrogenase, and peptidyl-prolyl isomerase. Surprisingly, PCNA is also associated with six glycolytic enzymes that are involved in the regulation of steps 4-9 in the glycolysis pathway. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation

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

Corbin, Cyrielle; Drouet, Samantha; Markulin, Lucija

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8' lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (-)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset ofmore » genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8' linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (-)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.« less

A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.

PubMed

Corbin, Cyrielle; Drouet, Samantha; Markulin, Lucija; Auguin, Daniel; Lainé, Éric; Davin, Laurence B; Cort, John R; Lewis, Norman G; Hano, Christophe

2018-05-01

Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8' lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (-)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8' linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (-)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.

Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants

PubMed Central

Jacobson, Samuel G.; Cideciyan, Artur V.; Peshenko, Igor V.; Sumaroka, Alexander; Olshevskaya, Elena V.; Cao, Lihui; Schwartz, Sharon B.; Roman, Alejandro J.; Olivares, Melani B.; Sadigh, Sam; Yau, King-Wai; Heon, Elise; Stone, Edwin M.; Dizhoor, Alexander M.

2013-01-01

The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months—37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo. We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1. PMID:23035049

[Two cases of personal identification from dental information].

PubMed

Yamaguchi, T; Yamada, Y; Ohtani, S; Kogure, T; Nagao, M; Takatori, T; Ohira, H; Yamamoto, I; Watanabe, A

1997-08-01

We describe two cases in which unknown bodies were positively identified from dental information and biochemical examination using tooth materials. In one case, a charred body was positively identified with little effort by comparison of antemortem dental records (dental chart and dental X-ray film) with postmortem data. In the other case, although the unknown individual had dental treatment, the police were unable to obtain the antemortem dental records of the victim. We then conducted biochemical analysis of teeth, facilitating personal identification using DNA analysis and age estimation based on aspartic acid racemization. The mutation obtained from the sequence of mtDNA and the genotypes of HLADQ alpha, HPRTB and ABO blood groups including the data for estimated age supported the kinship between the unknown individual and his mother. The data for maternally inherited mtDNA were of great importance in this case, since it was possible to obtain DNA from the mother. Dental identification in one of the most accurate methods of personal identification if suitable antemortem records are available. In the absence of such records, biochemical analysis of teeth also makes it possible to increase the probability of correct personal identification.

Known structure, unknown function: An inquiry-based undergraduate biochemistry laboratory course.

PubMed

Gray, Cynthia; Price, Carol W; Lee, Christopher T; Dewald, Alison H; Cline, Matthew A; McAnany, Charles E; Columbus, Linda; Mura, Cameron

2015-01-01

Undergraduate biochemistry laboratory courses often do not provide students with an authentic research experience, particularly when the express purpose of the laboratory is purely instructional. However, an instructional laboratory course that is inquiry- and research-based could simultaneously impart scientific knowledge and foster a student's research expertise and confidence. We have developed a year-long undergraduate biochemistry laboratory curriculum wherein students determine, via experiment and computation, the function of a protein of known three-dimensional structure. The first half of the course is inquiry-based and modular in design; students learn general biochemical techniques while gaining preparation for research experiments in the second semester. Having learned standard biochemical methods in the first semester, students independently pursue their own (original) research projects in the second semester. This new curriculum has yielded an improvement in student performance and confidence as assessed by various metrics. To disseminate teaching resources to students and instructors alike, a freely accessible Biochemistry Laboratory Education resource is available at http://biochemlab.org. © 2015 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.

Solving the puzzles of cutin and suberin polymer biosynthesis.

PubMed

Beisson, Fred; Li-Beisson, Yonghua; Pollard, Mike

2012-06-01

Cutin and suberin are insoluble lipid polymers that provide critical barrier functions to the cell wall of certain plant tissues, including the epidermis, endodermis and periderm. Genes that are specific to the biosynthesis of cutins and/or aliphatic suberins have been identified, mainly in Arabidopsis thaliana. They notably encode acyltransferases, oxidases and transporters, which may have either well-defined or more debatable biochemical functions. However, despite these advances, important aspects of cutin and suberin synthesis remain obscure. Central questions include whether fatty acyl monomers or oligomers are exported, and the extent of extracellular assembly and attachment to the cell wall. These issues are reviewed. Greater emphasis on chemistry and biochemistry will be required to solve these unknowns and link structure with function. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structure-informed insights for NLR functioning in plant immunity.

PubMed

Sukarta, Octavina C A; Slootweg, Erik J; Goverse, Aska

2016-08-01

To respond to foreign invaders, plants have evolved a cell autonomous multilayered immune system consisting of extra- and intracellular immune receptors. Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) mediate recognition of pathogen effectors inside the cell and trigger a host specific defense response, often involving controlled cell death. NLRs consist of a central nucleotide-binding domain, which is flanked by an N-terminal CC or TIR domain and a C-terminal leucine-rich repeat domain (LRR). These multidomain proteins function as a molecular switch and their activity is tightly controlled by intra and inter-molecular interactions. In contrast to metazoan NLRs, the structural basis underlying NLR functioning as a pathogen sensor and activator of immune responses in plants is largely unknown. However, the first crystal structures of a number of plant NLR domains were recently obtained. In addition, biochemical and structure-informed analyses revealed novel insights in the cooperation between NLR domains and the formation of pre- and post activation complexes, including the coordinated activity of NLR pairs as pathogen sensor and executor of immune responses. Moreover, the discovery of novel integrated domains underscores the structural diversity of NLRs and provides alternative models for how these immune receptors function in plants. In this review, we will highlight these recent advances to provide novel insights in the structural, biochemical and molecular aspects involved in plant NLR functioning. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

The use of biochemical methods in extraterrestrial life detection

NASA Astrophysics Data System (ADS)

McDonald, Gene

2006-08-01

Instrument development for in situ extraterrestrial life detection focuses primarily on the ability to distinguish between biological and non-biological material, mostly through chemical analysis for potential biosignatures (e.g., biogenic minerals, enantiomeric excesses). In constrast, biochemical analysis techniques commonly applied to Earth life focus primarily on the exploration of cellular and molecular processes, not on the classification of a given system as biological or non-biological. This focus has developed because of the relatively large functional gap between life and non-life on Earth today. Life on Earth is very diverse from an environmental and physiological point of view, but is highly conserved from a molecular point of view. Biochemical analysis techniques take advantage of this similarity of all terrestrial life at the molecular level, particularly through the use of biologically-derived reagents (e.g., DNA polymerases, antibodies), to enable analytical methods with enormous sensitivity and selectivity. These capabilities encourage consideration of such reagents and methods for use in extraterrestrial life detection instruments. The utility of this approach depends in large part on the (unknown at this time) degree of molecular compositional differences between extraterrestrial and terrestrial life. The greater these differences, the less useful laboratory biochemical techniques will be without significant modification. Biochemistry and molecular biology methods may need to be "de-focused" in order to produce instruments capable of unambiguously detecting a sufficiently wide range of extraterrestrial biochemical systems. Modern biotechnology tools may make that possible in some cases.

Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family

PubMed Central

Sinha, Sangita; Rappu, Pekka; Lange, S. C.; Mäntsälä, Pekka; Zalkin, Howard; Smith, Janet L.

1999-01-01

The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-Å crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins. PMID:10557275

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

PubMed

Wang, Nan; Zhang, Yeting; Gedvilaite, Erika; Loh, Jui Wan; Lin, Timothy; Liu, Xiuping; Liu, Chang-Gong; Kumar, Dibyendu; Donnelly, Robert; Raymond, Kimiyo; Schuchman, Edward H; Sleat, David E; Lobel, Peter; Xing, Jinchuan

2017-11-01

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect. © 2017 Wiley Periodicals, Inc.

Barnacle cement: a polymerization model based on evolutionary concepts

PubMed Central

Dickinson, Gary H.; Vega, Irving E.; Wahl, Kathryn J.; Orihuela, Beatriz; Beyley, Veronica; Rodriguez, Eva N.; Everett, Richard K.; Bonaventura, Joseph; Rittschof, Daniel

2009-01-01

Summary Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. PMID:19837892

Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of DRR206 and phytoalexin pathway localization.

PubMed

Seneviratne, Herana Kamal; Dalisay, Doralyn S; Kim, Kye-Won; Moinuddin, Syed G A; Yang, Hong; Hartshorn, Christopher M; Davin, Laurence B; Lewis, Norman G

2015-05-01

Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being pinoresinol monoglucoside. Interestingly, both pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and pinoresinol monoglucoside function in the overall phytoalexin responses. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

PubMed Central

Katz, Maximiliano J.; Acevedo, Julieta M.; Loenarz, Christoph; Galagovsky, Diego; Liu-Yi, Phebee; Pérez-Pepe, Marcelo; Thalhammer, Armin; Sekirnik, Rok; Ge, Wei; Melani, Mariana; Thomas, María G.; Simonetta, Sergio; Boccaccio, Graciela L.; Schofield, Christopher J.; Cockman, Matthew E.; Ratcliffe, Peter J.; Wappner, Pablo

2014-01-01

Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase. PMID:24550463

Biogenic Magnetite and EMF Effects

NASA Astrophysics Data System (ADS)

Kirschvink, Joseph L.

1996-03-01

Magnetite biomineralization is a genetically-controlled biochemical process through which organisms make perfect ferrimagnetic crystals, usually of single magnetic domain size. This process is an ancient one, having evolved about 2 billion years ago in the magnetotactic bacteria, and presumably was incorporated in the genome of higher organisms, including humans. During this time, DNA replication, protein synthesis, and many other biochemical processes have functioned in the presence of strong static fields of up to 400 mT adjacent to these magnetosomes without any obvious deleterious effects. Recent behavioral experiments using short but strong magnetic pulses in honeybees and birds demonstrates that ferromagnetic materials are involved in the sensory transduction of geomagnetic field information to the nervous system, and both behavioral and direct electrophysiological experiments indicate sensitivity thresholds to DC magnetic fields down to a few nT. However, far more biogenic magnetite is present in animal tissues than is needed for magnetoreception, and the biological function of this extra material is unknown. The presence of ferromagnetic materials in biological systems could provide physical transduction mechanisms for ELF magnetic fields, as well for microwave radiation in the .5 to 10 GHz band where magnetite has its peak ferromagnetic resonance. Elucidation of the cellular ultrastructure and biological function(s) of magnetite might help resolve the question of whether anthropogenic EMFs can cause deleterious biological effects. This work has been supported by grants from the NIH and EPRI.

Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

PubMed Central

Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

2013-01-01

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

Actinomyces cardiffensis sp. nov. from Human Clinical Sources

PubMed Central

Hall, Val; Collins, Mattew D.; Hutson, Roger; Falsen, Enevold; Duerden, Brian I.

2002-01-01

Eight strains of a previously undescribed catalase-negative Actinomyces-like bacterium were recovered from human clinical specimens. The morphological and biochemical characteristics of the isolates were consistent with their assignment to the genus Actinomyces, but they did not appear to correspond to any recognized species. 16S rRNA gene sequence analysis showed the organisms represent a hitherto unknown species within the genus Actinomyces related to, albeit distinct from, a group of species which includes Actinomyces turicensis and close relatives. Based on biochemical and molecular genetic evidence, it is proposed that the unknown isolates from human clinical sources be classified as a new species, Actinomyces cardiffensis sp. nov. The type strain of Actinomyces cardiffensis is CCUG 44997T. PMID:12202588

Known structure, unknown function: An inquiry‐based undergraduate biochemistry laboratory course

PubMed Central

Gray, Cynthia; Price, Carol W.; Lee, Christopher T.; Dewald, Alison H.; Cline, Matthew A.; McAnany, Charles E.

2015-01-01

Abstract Undergraduate biochemistry laboratory courses often do not provide students with an authentic research experience, particularly when the express purpose of the laboratory is purely instructional. However, an instructional laboratory course that is inquiry‐ and research‐based could simultaneously impart scientific knowledge and foster a student's research expertise and confidence. We have developed a year‐long undergraduate biochemistry laboratory curriculum wherein students determine, via experiment and computation, the function of a protein of known three‐dimensional structure. The first half of the course is inquiry‐based and modular in design; students learn general biochemical techniques while gaining preparation for research experiments in the second semester. Having learned standard biochemical methods in the first semester, students independently pursue their own (original) research projects in the second semester. This new curriculum has yielded an improvement in student performance and confidence as assessed by various metrics. To disseminate teaching resources to students and instructors alike, a freely accessible Biochemistry Laboratory Education resource is available at http://biochemlab.org. © 2015 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 43(4):245–262, 2015. PMID:26148241

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE PAGES

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

2014-11-20

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

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

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

Molecular-genetic characterization and rescue of a TSFM mutation causing childhood-onset ataxia and nonobstructive cardiomyopathy

PubMed Central

Emperador, Sonia; Bayona-Bafaluy, M Pilar; Fernández-Marmiesse, Ana; Pineda, Mercedes; Felgueroso, Blanca; López-Gallardo, Ester; Artuch, Rafael; Roca, Iria; Ruiz-Pesini, Eduardo; Couce, María Luz; Montoya, Julio

2017-01-01

Oxidative phosphorylation dysfunction has been found in many different disorders. This biochemical pathway depends on mitochondrial protein synthesis. Thus, mutations in components of the mitochondrial translation system can be responsible for some of these pathologies. We identified a new homozygous missense mutation in the mitochondrial translation elongation factor Ts gene in a patient suffering from slowly progressive childhood ataxia and hypertrophic cardiomyopathy. Using cell, biochemical and molecular-genetic protocols, we confirm it as the etiologic factor of this phenotype. Moreover, as an important functional confirmation, we rescued the normal molecular phenotype by expression of the wild-type TSFM cDNA in patient's fibroblasts. Different TSFM mutations can produce the same or very different clinical phenotypes, going from abortions to moderately severe presentations. On the other hand, the same TSFM mutation can also produce same or different phenotypes within the same range of presentations, therefore suggesting the involvement of unknown factors. PMID:27677415

The Enzyme Function Initiative†

PubMed Central

Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

2011-01-01

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID:21999478

The Enzyme Function Initiative.

PubMed

Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

2011-11-22

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts. © 2011 American Chemical Society

Laboratory Practical Exams in the Biochemistry Lab Course.

ERIC Educational Resources Information Center

Robyt, John F.; White, Bernard J.

1990-01-01

Described are the composition, design, administration, and evaluation of practical examinations. A table of the composition of biochemical unknowns for analysis in practical examinations is included. (CW)

DOR undergoes nucleo-cytoplasmic shuttling, which involves passage through the nucleolus.

PubMed

Mauvezin, Caroline; Sancho, Ana; Ivanova, Saska; Palacin, Manuel; Zorzano, Antonio

2012-09-21

DOR is a bi-functional protein that regulates transcription and enhances starvation-induced autophagy. While autophagy has been mostly described as a stress-response mechanism, cells also need autophagy to maintain homeostasis in basal conditions. However, the mechanisms regulating basal autophagy still remain unknown. Our results show that DOR acts in basal autophagy. Indeed, DOR already undergoes nucleo-cytoplasmic shuttling in basal conditions and, surprisingly, DOR exits continuously the nucleus and traverses the nucleolus. However, the nucleolus integrity is not essential for both DOR nucleo-cytoplasmic shuttling and DOR function on basal autophagy. Taken together, we propose that DOR exit from the nucleus is essential for basal autophagy stimulation even under nucleolus disruption. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation.

PubMed

Toyama, H; Anthony, C; Lidstrom, M E

1998-09-01

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

Exploration of Uncharted Regions of the Protein Universe

PubMed Central

Jaroszewski, Lukasz; Li, Zhanwen; Krishna, S. Sri; Bakolitsa, Constantina; Wooley, John; Deacon, Ashley M.; Wilson, Ian A.; Godzik, Adam

2009-01-01

The genome projects have unearthed an enormous diversity of genes of unknown function that are still awaiting biological and biochemical characterization. These genes, as most others, can be grouped into families based on sequence similarity. The PFAM database currently contains over 2,200 such families, referred to as domains of unknown function (DUF). In a coordinated effort, the four large-scale centers of the NIH Protein Structure Initiative have determined the first three-dimensional structures for more than 250 of these DUF families. Analysis of the first 248 reveals that about two thirds of the DUF families likely represent very divergent branches of already known and well-characterized families, which allows hypotheses to be formulated about their biological function. The remainder can be formally categorized as new folds, although about one third of these show significant substructure similarity to previously characterized folds. These results infer that, despite the enormous increase in the number and the diversity of new genes being uncovered, the fold space of the proteins they encode is gradually becoming saturated. The previously unexplored sectors of the protein universe appear to be primarily shaped by extreme diversification of known protein families, which then enables organisms to evolve new functions and adapt to particular niches and habitats. Notwithstanding, these DUF families still constitute the richest source for discovery of the remaining protein folds and topologies. PMID:19787035

An Integrated Encyclopedia of DNA Elements in the Human Genome

PubMed Central

2012-01-01

Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

Selenium biochemistry and its role for human health.

PubMed

Roman, Marco; Jitaru, Petru; Barbante, Carlo

2014-01-01

Despite its very low level in humans, selenium plays an important and unique role among the (semi)metal trace essential elements because it is the only one for which incorporation into proteins is genetically encoded, as the constitutive part of the 21st amino acid, selenocysteine. Twenty-five selenoproteins have been identified so far in the human proteome. The biological functions of some of them are still unknown, whereas for others there is evidence for a role in antioxidant defence, redox state regulation and a wide variety of specific metabolic pathways. In relation to these functions, the selenoproteins emerged in recent years as possible biomarkers of several diseases such as diabetes and several forms of cancer. Comprehension of the selenium biochemical pathways under normal physiological conditions is therefore an important requisite to elucidate its preventing/therapeutic effect for human diseases. This review summarizes the most recent findings on the biochemistry of active selenium species in humans, and addresses the latest evidence on the link between selenium intake, selenoproteins functionality and beneficial health effects. Primary emphasis is given to the interpretation of biochemical mechanisms rather than epidemiological/observational data. In this context, the review includes the following sections: (1) brief introduction; (2) general nutritional aspects of selenium; (3) global view of selenium metabolic routes; (4) detailed characterization of all human selenoproteins; (5) detailed discussion of the relation between selenoproteins and a variety of human diseases.

In situ metabolomic- and transcriptomic-profiling of the host-associated cyanobacteria Prochloron and Acaryochloris marina.

PubMed

Behrendt, Lars; Raina, Jean-Baptiste; Lutz, Adrian; Kot, Witold; Albertsen, Mads; Halkjær-Nielsen, Per; Sørensen, Søren J; Larkum, Anthony Wd; Kühl, Michael

2017-10-31

The tropical ascidian Lissoclinum patella hosts two enigmatic cyanobacteria: (1) the photoendosymbiont Prochloron spp., a producer of valuable bioactive compounds and (2) the chlorophyll-d containing Acaryochloris spp., residing in the near-infrared enriched underside of the animal. Despite numerous efforts, Prochloron remains uncultivable, restricting the investigation of its biochemical potential to cultivation-independent techniques. Likewise, in both cyanobacteria, universally important parameters on light-niche adaptation and in situ photosynthetic regulation are unknown. Here we used genome sequencing, transcriptomics and metabolomics to investigate the symbiotic linkage between host and photoendosymbiont and simultaneously probed the transcriptional response of Acaryochloris in situ. During high light, both cyanobacteria downregulate CO 2 fixing pathways, likely a result of O 2 photorespiration on the functioning of RuBisCO, and employ a variety of stress-quenching mechanisms, even under less stressful far-red light (Acaryochloris). Metabolomics reveals a distinct biochemical modulation between Prochloron and L. patella, including noon/midnight-dependent signatures of amino acids, nitrogenous waste products and primary photosynthates. Surprisingly, Prochloron constitutively expressed genes coding for patellamides, that is, cyclic peptides of great pharmaceutical value, with yet unknown ecological significance. Together these findings shed further light on far-red-driven photosynthesis in natural consortia, the interplay of Prochloron and its ascidian partner in a model chordate photosymbiosis and the uncultivability of Prochloron.The ISME Journal advance online publication, 31 October 2017; doi:10.1038/ismej.2017.192.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

PubMed

Lee-Barthel, Ann; Baar, Keith; West, Daniel W D

2017-06-11

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

PubMed Central

Lee-Barthel, Ann; Baar, Keith; West, Daniel W. D.

2017-01-01

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration. PMID:28654031

Comparative Metagenomics of Cellulose- and Poplar Hydrolysate-Degrading Microcosms from Gut Microflora of the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus) after Long-Term Enrichment

PubMed Central

Wong, Mabel T.; Wang, Weijun; Couturier, Marie; Razeq, Fakhria M.; Lombard, Vincent; Lapebie, Pascal; Edwards, Elizabeth A.; Terrapon, Nicolas; Henrissat, Bernard; Master, Emma R.

2017-01-01

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization. PMID:29326667

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

PubMed

Jiang, Tianyu; Liu, Minhao; Wu, Jianping; Shi, Yigong

2016-02-23

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

Extraordinary Structured Noncoding RNAs Revealed by Bacterial Metagenome Analysis

PubMed Central

Weinberg, Zasha; Perreault, Jonathan; Meyer, Michelle M.; Breaker, Ronald R.

2012-01-01

Estimates of the total number of bacterial species1-3 suggest that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6-10 of genomic DNA from bacteria commonly identify novel noncoding RNAs (ncRNAs)10-12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, suggesting that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space. PMID:19956260

LKB1 and lung cancer: more than the usual suspects.

PubMed

Shah, Usman; Sharpless, Norman E; Hayes, D Neil

2008-05-15

Often, the problem in cancer research is figuring out how a gene or pathway works in regulating cellular transformation. The question of what RAS activates or PTEN inhibits have been classic dilemmas of modern cancer biology. In these cases, biochemical and genetic studies have provided us with a fairly clear picture of the cancer relevant functions of these genes. For LKB1, a more recently identified human tumor suppressor gene, however, the problem is different. This serine-threonine kinase that is conserved from yeast to mammals seems to play a role in many diverse cellular pathways. Therefore, although elegant functional and genetic approaches have established critical roles for LKB1 in the regulation of metabolism, motility, polarity, and the cell cycle, the role(s) responsible for its true tumor suppressor function(s) is unknown. One is reminded of an Agatha Christie murder mystery where nearly every character in the book has reason to be suspected of committing the crime-there are too many suspects for how LKB1 might repress lung cancer.

Sirtuins, Bioageing, and Cancer

PubMed Central

McGuinness, D.; McGuinness, D. H.; McCaul, J. A.; Shiels, P. G.

2011-01-01

The Sirtuins are a family of orthologues of yeast Sir2 found in a wide range of organisms from bacteria to man. They display a high degree of conservation between species, in both sequence and function, indicative of their key biochemical roles. Sirtuins are heavily implicated in cell cycle, cell division, transcription regulation, and metabolism, which places the various family members at critical junctures in cellular metabolism. Typically, Sirtuins have been implicated in the preservation of genomic stability and in the prolongation of lifespan though many of their target interactions remain unknown. Sirtuins play key roles in tumourigenesis, as some have tumour-suppressor functions and others influence tumours through their control of the metabolic state of the cell. Their links to ageing have also highlighted involvement in various age-related and degenerative diseases. Here, we discuss the current understanding of the role of Sirtuins in age-related diseases while taking a closer look at their roles and functions in maintaining genomic stability and their influence on telomerase and telomere function. PMID:21766030

Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response.

PubMed

Rice, Gillian I; Bond, Jacquelyn; Asipu, Aruna; Brunette, Rebecca L; Manfield, Iain W; Carr, Ian M; Fuller, Jonathan C; Jackson, Richard M; Lamb, Teresa; Briggs, Tracy A; Ali, Manir; Gornall, Hannah; Couthard, Lydia R; Aeby, Alec; Attard-Montalto, Simon P; Bertini, Enrico; Bodemer, Christine; Brockmann, Knut; Brueton, Louise A; Corry, Peter C; Desguerre, Isabelle; Fazzi, Elisa; Cazorla, Angels Garcia; Gener, Blanca; Hamel, Ben C J; Heiberg, Arvid; Hunter, Matthew; van der Knaap, Marjo S; Kumar, Ram; Lagae, Lieven; Landrieu, Pierre G; Lourenco, Charles M; Marom, Daphna; McDermott, Michael F; van der Merwe, William; Orcesi, Simona; Prendiville, Julie S; Rasmussen, Magnhild; Shalev, Stavit A; Soler, Doriette M; Shinawi, Marwan; Spiegel, Ronen; Tan, Tiong Y; Vanderver, Adeline; Wakeling, Emma L; Wassmer, Evangeline; Whittaker, Elizabeth; Lebon, Pierre; Stetson, Daniel B; Bonthron, David T; Crow, Yanick J

2009-07-01

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response.

Characterization of breakpoint cluster region kinase and SH2-binding activities.

PubMed

Afar, D E; Witte, O N

1995-01-01

BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities.

Mutations involved in Aicardi-Goutières syndrome implicate SAMHD1 as regulator of the innate immune response

PubMed Central

Rice, Gillian I; Bond, Jacquelyn; Asipu, Aruna; Brunette, Rebecca L; Manfield, Iain W; Carr, Ian M; Fuller, Jonathan C; Jackson, Richard M; Lamb, Teresa; Briggs, Tracy A; Ali, Manir; Gornall, Hannah; Couthard, Lydia R; Aeby, Alec; Attard-Montalto, Simon P; Bertini, Enrico; Bodemer, Christine; Brockmann, Knut; Brueton, Louise A; Corry, Peter C; Desguerre, Isabelle; Fazzi, Elisa; Cazorla, Angels Garcia; Gener, Blanca; Hamel, Ben C J; Heiberg, Arvid; Hunter, Matthew; van der Knaap, Marjo S; Kumar, Ram; Lagae, Lieven; Landrieu, Pierre G; Lourenco, Charles M; Marom, Daphna; McDermott, Michael F; van der Merwe, William; Orcesi, Simona; Prendiville, Julie S; Rasmussen, Magnhild; Shalev, Stavit A; Soler, Doriette M; Shinawi, Marwan; Spiegel, Ronen; Tan, Tiong Y; Vanderver, Adeline; Wakeling, Emma L; Wassmer, Evangeline; Whittaker, Elizabeth; Lebon, Pierre; Stetson, Daniel B; Bonthron, David T; Crow, Yanick J

2014-01-01

Aicardi-Goutières syndrome is a mendelian mimic of congenital infection and also shows overlap with systemic lupus erythematosus at both a clinical and biochemical level. The recent identification of mutations in TREX1 and genes encoding the RNASEH2 complex and studies of the function of TREX1 in DNA metabolism have defined a previously unknown mechanism for the initiation of autoimmunity by interferon-stimulatory nucleic acid. Here we describe mutations in SAMHD1 as the cause of AGS at the AGS5 locus and present data to show that SAMHD1 may act as a negative regulator of the cell-intrinsic antiviral response. PMID:19525956

Renal dopamine containing nerves. What is their functional significance?

PubMed

DiBona, G F

1990-06-01

Biochemical and morphological studies indicate that there are nerves within the kidney that contain dopamine and that various structures within the kidney contain dopamine receptors. However, the functional significance of these renal dopamine containing nerves in relation to renal dopamine receptors is unknown. The functional significance could be defined by demonstrating that an alteration in one or more renal functions occurring in response to reflex or electrical activation of efferent renal nerves is dependent on release of dopamine as the neurotransmitter from the renal nerve terminals acting on renal dopamine receptors. Thus, the hypothesis becomes: reflex or electrical activation of efferent renal nerves causes alterations in renal function (eg, renal blood flow, water and solute handling) that are inhibited by specific and selective dopamine receptor antagonists. As reviewed herein, the published experimental data do not support the hypothesis. Therefore, the view that alterations in one or more renal functions occurring in response to reflex or electrical activation of efferent renal nerves are dependent on release of dopamine as the neurotransmitter from the renal nerve terminals acting on renal dopamine receptors remains unproven.

Streptococcus caprae sp. nov., isolated from Iberian ibex (Capra pyrenaica hispanica).

PubMed

Vela, A I; Mentaberre, G; Lavín, S; Domínguez, L; Fernández-Garayzábal, J F

2016-01-01

Biochemical and molecular genetic studies were performed on a novel Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from tonsil samples of two Iberian ibexes. The micro-organism was identified as a streptococcal species based on its cellular, morphological and biochemical characteristics. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from ibex was Streptococcus porci 2923-03T (96.6 % 16S rRNA gene sequence similarity). Analysis based on rpoB and sodA gene sequences revealed sequence similarity values lower than 86.0 and 83.8 %, respectively, from the type strains of recognized Streptococcus species. The novel bacterial isolate was distinguished from Streptococcus porci and other Streptococcus species using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as representing a novel species of the genus Streptococcus, for which the name Streptococcus caprae sp. nov. is proposed. The type strain is DICM07-02790-1CT ( = CECT 8872T = CCUG 67170T).

The Molecular Basis of Wound Healing Processes Induced by Lithospermi Radix: A Proteomics and Biochemical Analysis

PubMed Central

Hsiao, Chia-Yen; Tsai, Tung-Hu; Chak, Kin-Fu

2012-01-01

Lithospermi Radix (LR) is an effective traditional Chinese herb in various types of wound healing; however, its mechanism of action remains unknown. A biochemical and proteomic platform was generated to explore the biological phenomena associated with LR and its active component shikonin. We found that both LR ethanol extracts and shikonin are able to promote cell proliferation by up to 25%. The results of proteomic analysis revealed that twenty-two differentially expressed proteins could be identified when fibroblast cells were treated with LR or shikonin. The functions of those proteins are associated with antioxidant activity, antiapoptosis activity, the regulation of cell mobility, the secretion of collagen, the removal of abnormal proteins, and the promotion of cell proliferation, indicating that the efficacy of LR in wound healing may be derived from a synergistic effect on a number of factors induced by the herbal medicine. Furthermore, an animal model confirmed that LR is able to accelerate wound healing on the flank back of the SD rats. Together these findings help to pinpoint the molecular basis of wound healing process induced by LR. PMID:23024692

Cooperative ethylene receptor signaling

PubMed Central

Liu, Qian; Wen, Chi-Kuang

2012-01-01

The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses by multiple ethylene receptors has yet to be fully addressed. Nevertheless, the ethylene receptor signal strength can be reflected by degrees in alteration of various ethylene response phenotypes and in expression levels of ethylene-inducible genes. This mini-review highlights studies that have advanced our understanding of cooperative ethylene receptor signaling. PMID:22827938

Rapid analytical determination of glutaraldehyde concentrations

NASA Technical Reports Server (NTRS)

Frigerio, N. A.; Shaw, M. H.

1971-01-01

Technique utilizes the iodimetric procedure which adds unknown excess of bisulfite to glutaraldehyde /GA/ then titrates unreacted bisulfite with standard iodine isotope to determine GA concentrations. Technique may interest microscopists, food researchers, biochemical or medical laboratories, and drug manufacturers.

An Alternative Approach to "Identification of Unknowns": Designing a Protocol to Verify the Identities of Nitrogen Fixing Bacteria.

PubMed

Martinez-Vaz, Betsy M; Denny, Roxanne; Young, Nevin D; Sadowsky, Michael J

2015-12-01

Microbiology courses often include a laboratory activity on the identification of unknown microbes. This activity consists of providing students with microbial cultures and running biochemical assays to identify the organisms. This approach lacks molecular techniques such as sequencing of genes encoding 16S rRNA, which is currently the method of choice for identification of unknown bacteria. A laboratory activity was developed to teach students how to identify microorganisms using 16S rRNA polymerase chain reaction (PCR) and validate microbial identities using biochemical techniques. We hypothesized that designing an experimental protocol to confirm the identity of a bacterium would improve students' knowledge of microbial identification techniques and the physiological characteristics of bacterial species. Nitrogen-fixing bacteria were isolated from the root nodules of Medicago truncatula and prepared for 16S rRNA PCR analysis. Once DNA sequencing revealed the identity of the organisms, the students designed experimental protocols to verify the identity of rhizobia. An assessment was conducted by analyzing pre- and posttest scores and by grading students' verification protocols and presentations. Posttest scores were higher than pretest scores at or below p = 0.001. Normalized learning gains (G) showed an improvement of students' knowledge of microbial identification methods (LO4, G = 0.46), biochemical properties of nitrogen-fixing bacteria (LO3, G = 0.45), and the events leading to the establishment of nitrogen-fixing symbioses (LO1&2, G = 0.51, G = 0.37). An evaluation of verification protocols also showed significant improvement with a p value of less than 0.001.

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

PubMed

Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

2014-09-02

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

PubMed

Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

2014-01-21

Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Modulatory Effects of Dietary Amino Acids on Neurodegenerative Diseases.

PubMed

Rajagopal, Senthilkumar; Sangam, Supraj Raja; Singh, Shubham; Joginapally, Venkateswara Rao

2016-01-01

Proteins are playing a vital role in maintaining the cellular integrity and function, as well as for brain cells. Protein intake and supplementation of individual amino acids can affect the brain functioning and mental health, and many of the neurotransmitters in the brain are made from amino acids. The amino acid supplementation has been found to reduce symptoms, as they are converted into neurotransmitters which in turn extenuate the mental disorders. The biosynthesis of amino acids in the brain is regulated by the concentration of amino acids in plasma. The brain diseases such as depression, bipolar disorder, schizophrenia, obsessive-compulsive disorder (OCD), and Alzheimer's (AD), Parkinson's (PD), and Huntington's diseases (HD) are the most common mental disorders that are currently widespread in numerous countries. The intricate biochemical and molecular machinery contributing to the neurological disorders is still unknown, and in this chapter, we revealed the involvement of dietary amino acids on neurological diseases.

Selection on Network Dynamics Drives Differential Rates of Protein Domain Evolution

PubMed Central

Mannakee, Brian K.; Gutenkunst, Ryan N.

2016-01-01

The long-held principle that functionally important proteins evolve slowly has recently been challenged by studies in mice and yeast showing that the severity of a protein knockout only weakly predicts that protein’s rate of evolution. However, the relevance of these studies to evolutionary changes within proteins is unknown, because amino acid substitutions, unlike knockouts, often only slightly perturb protein activity. To quantify the phenotypic effect of small biochemical perturbations, we developed an approach to use computational systems biology models to measure the influence of individual reaction rate constants on network dynamics. We show that this dynamical influence is predictive of protein domain evolutionary rate within networks in vertebrates and yeast, even after controlling for expression level and breadth, network topology, and knockout effect. Thus, our results not only demonstrate the importance of protein domain function in determining evolutionary rate, but also the power of systems biology modeling to uncover unanticipated evolutionary forces. PMID:27380265

Calcium channels in chicken sperm regulate motility and the acrosome reaction.

PubMed

Nguyen, Thi Mong Diep; Duittoz, Anne; Praud, Christophe; Combarnous, Yves; Blesbois, Elisabeth

2016-05-01

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction. © 2016 Federation of European Biochemical Societies.

Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease.

PubMed

Raththagala, Madushi; Brewer, M Kathryn; Parker, Matthew W; Sherwood, Amanda R; Wong, Brian K; Hsu, Simon; Bridges, Travis M; Paasch, Bradley C; Hellman, Lance M; Husodo, Satrio; Meekins, David A; Taylor, Adam O; Turner, Benjamin D; Auger, Kyle D; Dukhande, Vikas V; Chakravarthy, Srinivas; Sanz, Pascual; Woods, Virgil L; Li, Sheng; Vander Kooi, Craig W; Gentry, Matthew S

2015-01-22

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapamycin mitigates erythrocyte membrane transport functions and oxidative stress during aging in rats.

PubMed

Singh, Abhishek Kumar; Singh, Sandeep; Garg, Geetika; Rizvi, Syed Ibrahim

2018-02-01

Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (-SH), sialic acid (SA) and intracellular calcium ion [Ca 2+ ]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na + /K + -ATPase (NKA) and Ca 2+ -ATPase (PMCA), and levels of -SH and SA was observed along with a simultaneous induction in Na + /H + exchanger (NHE) activity and levels of [Ca 2+ ]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.

Gibberellic Acid: A Key Phytohormone for Spikelet Fertility in Rice Grain Production.

PubMed

Kwon, Choon-Tak; Paek, Nam-Chon

2016-05-23

The phytohormone gibberellic acid (GA) has essential signaling functions in multiple processes during plant development. In the "Green Revolution", breeders developed high-yield rice cultivars that exhibited both semi-dwarfism and altered GA responses, thus improving grain production. Most studies of GA have concentrated on germination and cell elongation, but GA also has a pivotal role in floral organ development, particularly in stamen/anther formation. In rice, GA signaling plays an important role in spikelet fertility; however, the molecular genetic and biochemical mechanisms of GA in male fertility remain largely unknown. Here, we review recent progress in understanding the network of GA signaling and its connection with spikelet fertility, which is tightly associated with grain productivity in cereal crops.

The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor.

PubMed

Valdez, Benigno C; Henning, Dale; So, Rolando B; Dixon, Jill; Dixon, Michael J

2004-07-20

Treacher Collins syndrome (TCS) is an autosomal dominant disorder characterized by an abnormality of craniofacial development that arises during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Even though the genetic alterations causing TCS have been uncovered, the mechanism underlying its pathogenesis and the function of treacle remain unknown. Here, we show that treacle is involved in ribosomal DNA gene transcription by interacting with upstream binding factor (UBF). Immunofluorescence labeling shows treacle and UBF colocalize to specific nucleolar organizer regions and cosegregate within nucleolar caps of actinomycin d-treated HeLa cells. Biochemical analysis shows the association of treacle and UBF with chromatin. Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the two nucleolar phosphoproteins. Down-regulation of treacle expression using specific short interfering RNA results in inhibition of ribosomal DNA transcription and cell growth. A similar correlation is observed in Tcof(+/-) mouse embryos that exhibit craniofacial defects and growth retardation. Thus, treacle haploinsufficiency in TCS patients might result in abnormal development caused by inadequate ribosomal RNA production in the prefusion neural folds during the early stages of embryogenesis. The elucidation of a physiological function of treacle provides important information of relevance to the molecular dissection of the biochemical pathology of TCS.

The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor

PubMed Central

Valdez, Benigno C.; Henning, Dale; So, Rolando B.; Dixon, Jill; Dixon, Michael J.

2004-01-01

Treacher Collins syndrome (TCS) is an autosomal dominant disorder characterized by an abnormality of craniofacial development that arises during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Even though the genetic alterations causing TCS have been uncovered, the mechanism underlying its pathogenesis and the function of treacle remain unknown. Here, we show that treacle is involved in ribosomal DNA gene transcription by interacting with upstream binding factor (UBF). Immunofluorescence labeling shows treacle and UBF colocalize to specific nucleolar organizer regions and cosegregate within nucleolar caps of actinomycin d-treated HeLa cells. Biochemical analysis shows the association of treacle and UBF with chromatin. Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the two nucleolar phosphoproteins. Down-regulation of treacle expression using specific short interfering RNA results in inhibition of ribosomal DNA transcription and cell growth. A similar correlation is observed in Tcof+/- mouse embryos that exhibit craniofacial defects and growth retardation. Thus, treacle haploinsufficiency in TCS patients might result in abnormal development caused by inadequate ribosomal RNA production in the prefusion neural folds during the early stages of embryogenesis. The elucidation of a physiological function of treacle provides important information of relevance to the molecular dissection of the biochemical pathology of TCS. PMID:15249688

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

MetaMapR: pathway independent metabolomic network analysis incorporating unknowns.

PubMed

Grapov, Dmitry; Wanichthanarak, Kwanjeera; Fiehn, Oliver

2015-08-15

Metabolic network mapping is a widely used approach for integration of metabolomic experimental results with biological domain knowledge. However, current approaches can be limited by biochemical domain or pathway knowledge which results in sparse disconnected graphs for real world metabolomic experiments. MetaMapR integrates enzymatic transformations with metabolite structural similarity, mass spectral similarity and empirical associations to generate richly connected metabolic networks. This open source, web-based or desktop software, written in the R programming language, leverages KEGG and PubChem databases to derive associations between metabolites even in cases where biochemical domain or molecular annotations are unknown. Network calculation is enhanced through an interface to the Chemical Translation System, which allows metabolite identifier translation between >200 common biochemical databases. Analysis results are presented as interactive visualizations or can be exported as high-quality graphics and numerical tables which can be imported into common network analysis and visualization tools. Freely available at http://dgrapov.github.io/MetaMapR/. Requires R and a modern web browser. Installation instructions, tutorials and application examples are available at http://dgrapov.github.io/MetaMapR/. ofiehn@ucdavis.edu. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

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

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE PAGES

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; ...

2017-03-08

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Mitochondrial respiratory chain Complex I defects in Fanconi anemia complementation group A.

PubMed

Ravera, Silvia; Vaccaro, Daniele; Cuccarolo, Paola; Columbaro, Marta; Capanni, Cristina; Bartolucci, Martina; Panfoli, Isabella; Morelli, Alessandro; Dufour, Carlo; Cappelli, Enrico; Degan, Paolo

2013-10-01

Fanconi anemia (FA) is a rare and complex inherited blood disorder of the child. At least 15 genes are associated with the disease. The highest frequency of mutations belongs to groups A, C and G. Genetic instability and cytokine hypersensitivity support the selection of leukemic over non-leukemic stem cells. FA cellular phenotype is characterized by alterations in red-ox state, mitochondrial functionality and energy metabolism as reported in the past however a clear picture of the altered biochemical phenotype in FA is still elusive and the final biochemical defect(s) still unknown. Here we report an analysis of the respiratory fluxes in FANCA primary fibroblasts, lymphocytes and lymphoblasts. FANCA mutants show defective respiration through Complex I, diminished ATP production and metabolic sufferance with an increased AMP/ATP ratio. Respiration in FANCC mutants is normal. Treatment with N-acetyl-cysteine (NAC) restores oxygen consumption to normal level. Defective respiration in FANCA mutants appear correlated with the FA pro-oxidative phenotype which is consistent with the altered morphology of FANCA mitochondria. Electron microscopy measures indeed show profound alterations in mitochondrial ultrastructure and shape. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Biochemical transport modeling, estimation, and detection in realistic environments

NASA Astrophysics Data System (ADS)

Ortner, Mathias; Nehorai, Arye

2006-05-01

Early detection and estimation of the spread of a biochemical contaminant are major issues for homeland security applications. We present an integrated approach combining the measurements given by an array of biochemical sensors with a physical model of the dispersion and statistical analysis to solve these problems and provide system performance measures. We approximate the dispersion model of the contaminant in a realistic environment through numerical simulations of reflected stochastic diffusions describing the microscopic transport phenomena due to wind and chemical diffusion using the Feynman-Kac formula. We consider arbitrary complex geometries and account for wind turbulence. Localizing the dispersive sources is useful for decontamination purposes and estimation of the cloud evolution. To solve the associated inverse problem, we propose a Bayesian framework based on a random field that is particularly powerful for localizing multiple sources with small amounts of measurements. We also develop a sequential detector using the numerical transport model we propose. Sequential detection allows on-line analysis and detecting wether a change has occurred. We first focus on the formulation of a suitable sequential detector that overcomes the presence of unknown parameters (e.g. release time, intensity and location). We compute a bound on the expected delay before false detection in order to decide the threshold of the test. For a fixed false-alarm rate, we obtain the detection probability of a substance release as a function of its location and initial concentration. Numerical examples are presented for two real-world scenarios: an urban area and an indoor ventilation duct.

A systemic view on the distribution of diet-derived methanol and hepatic acetone in mice.

PubMed

Kistler, Martin; Muntean, Andreea; Höllriegl, Vera; Matuschek, Georg; Zimmermann, Ralf; Hoeschen, Christoph; de Angelis, Martin Hrabě; Rozman, Jan

2017-12-06

Volatile organic compounds (VOCs) from breath can successfully be used to diagnose disease-specific pathological alterations in metabolism. However, the exact origin and underlying biochemical pathways that could be mapped to VOC signatures are mainly unknown. There is a knowledge gap regarding the contribution of tissues, organs, the gut microbiome, and exogenous factors to the 'sum signal' from breath samples. Animal models for human disease such as mutant mice provide the possibility to reproduce genetic predisposition to disease, thereby allowing in-depth analysis of metabolic and biochemical functions. We hypothesized that breath VOCs can be traced back to origins and organ-specific metabolic functions by combining breath concentrations with systemic levels detected in different organs and biological media (breath, blood, feces and urine). For this we fed C57Bl/6N mice a grain-based chow or a purified low-fat diet, thereby modifying the emission of methanol in breath whereas acetone levels were unaffected. We then measured headspace concentrations of both VOCs in ex vivo samples of several biological media. Cecum content especially was identified as a likely source of systemic methanol, whereas the liver showed highest acetone concentrations. Our findings are a first step to the systemic mapping of VOC patterns to metabolic functions in mice because differences between VOCs could be traced to different sources in the body. As a future aim, different levels of so-called omics technologies (genomics, proteomics, metabolomics, and breathomics) could be mapped to metabolic pathways in multiple tissues, deepening our understanding of VOC metabolism and possibly leading to early non-invasive biomarkers for human pathologies.

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

PubMed

Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

2017-07-18

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

Muscle FBPase binds to cardiomyocyte mitochondria under glycogen synthase kinase-3 inhibition or elevation of cellular Ca2+ level.

PubMed

Gizak, Agnieszka; Pirog, Michal; Rakus, Dariusz

2012-01-02

A growing body of research suggests that fructose 1,6-bisphosphatase (FBPase) might be involved in regulation of cell mortality/survival. However, the precise role of FBPase in the process remains unknown. Here, we show for the first time that in HL-1 cardiomyocytes, inhibition of glycogen synthase kinase-3 results in translocation of FBPase to mitochondria. In vitro experiments demonstrate that FBPase reduces the rate of calcium-induced mitochondrial swelling, affects ATP synthesis and interacts with mitochondrial proteins involved in regulation of volume and energy homeostasis. We suggest that FBPase might be engaged in a regulation of cell survival by influencing mitochondrial function. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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.

THE USE OF GENE ARRAYS TO DETERMINE TEMPORAL GENE INDUCTION IN SHEEPSHEAD MINNOWS EXPOSED TO E2

EPA Science Inventory

Gene arrays provide a means to study differential gene expression in fish exposed to environmental estrogens by providing a "snapshot" of the genes expressed at a given time. Such array data may also uncover previously unknown biochemical pathways affected by estrogenic compounds...

The Python pit organ: imaging and immunocytochemical analysis of an extremely sensitive natural infrared detector.

PubMed

Grace, M S; Church, D R; Kelly, C T; Lynn, W F; Cooper, T M

1999-01-01

The Python infrared-sensitive pit organ is a natural infrared imager that combines high sensitivity, ambient temperature function, microscopic dimensions, and self-repair. We are investigating the spectral sensitivity and signal transduction process in snake infrared-sensitive neurons, neither of which is understood. For example, it is unknown whether infrared receptor neurons function on a thermal or a photic mechanism. We imaged pit organs in living Python molurus and Python regius using infrared-sensitive digital video cameras. Pit organs were significantly more absorptive and/or emissive than surrounding tissues in both 3-5 microns and 8-12 microns wavelength ranges. Pit organs exhibited greater absorption/emissivity in the 8-12 microns range than in the 3-5 microns range. To directly test the relationship between photoreceptors and pit organ infrared-sensitive neurons, we performed immunocytochemistry using antisera directed against retinal photoreceptor opsins. Retinal photoreceptors were labeled with antisera specific for retinal opsins, but these antisera failed to label terminals of infrared-sensitive neurons in the pit organ. Infrared-receptive neurons were also distinguished from retinal photoreceptors on the basis of their calcium-binding protein content. These results indicate that the pit organ absorbs infrared radiation in two major atmospheric transmission windows, one of which (8-12 microns) matches emission of targeted prey, and that infrared receptors are biochemically distinct from retinal photoreceptors. These results also provide the first identification of prospective biochemical components of infrared signal transduction in pit organ receptor neurons.

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism

PubMed Central

Faraldo-Gómez, José D.

2017-01-01

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation. PMID:29167180

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

PubMed

Liu, Na-Nv; Duan, Xiao-Lei; Ai, Xia; Yang, Yan-Tao; Li, Ming; Dou, Shuo-Xing; Rety, Stephane; Deprez, Eric; Xi, Xu-Guang

2015-10-15

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation*

PubMed Central

Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W.; Wanders, Ronald J. A.; Locasale, Jason W.; Lombard, David B.; de Boer, Vincent C. J.; Zhao, Yingming

2015-01-01

The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD−/− fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211

A novel pathogenesis of inflammatory bowel disease from the perspective of glyco-immunology.

PubMed

Shinzaki, Shinichiro; Iijima, Hideki; Fujii, Hironobu; Kamada, Yoshihiro; Naka, Tetsuji; Takehara, Tetsuo; Miyoshi, Eiji

2017-05-01

Oligosaccharide modifications play an essential role in various inflammatory diseases and cancers, but their pathophysiologic roles, especially in inflammation, are not clear. Inflammatory bowel disease (IBD) is an intractable chronic inflammatory disorder with an unknown aetiology, and the number of patients with IBD is increasing throughout the world. Certain types of immunosuppressant drugs, such as corticosteroids, are effective for IBD, suggesting that immune function is closely associated with the pathophysiology of IBD. Recent progress in the analysis of oligosaccharides revealed a role for oligosaccharides in intestinal inflammation based on both experimental models and human samples from IBD patients. Moreover, changes in the oligosaccharide structures on glycoproteins in the sera and tissue samples may serve as biomarkers of IBD. Here, we present current studies of IBD with regard to the immunologic aspects of glycobiology, suggesting a novel concept for IBD pathogenesis and the function of oligosaccharides on immune cells, termed "glyco-immunology". © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

PubMed

Xu, Qiang; Wang, Yanshu; Dabdoub, Alain; Smallwood, Philip M; Williams, John; Woods, Chad; Kelley, Matthew W; Jiang, Li; Tasman, William; Zhang, Kang; Nathans, Jeremy

2004-03-19

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

Selective loss of glycogen synthase kinase-3α in birds reveals distinct roles for GSK-3 isozymes in tau phosphorylation.

PubMed

Alon, Lina Tsaadon; Pietrokovski, Shmuel; Barkan, Shay; Avrahami, Limor; Kaidanovich-Beilin, Oksana; Woodgett, James R; Barnea, Anat; Eldar-Finkelman, Hagit

2011-04-20

Mammalian glycogen synthase kinase-3 (GSK-3), a critical regulator in neuronal signaling, cognition, and behavior, exists as two isozymes GSK-3α and GSK-3β. Their distinct biological functions remains largely unknown. Here, we examined the evolutionary significance of each of these isozymes. Surprisingly, we found that unlike other vertebrates that harbor both GSK-3 genes, the GSK-3α gene is missing in birds. GSK-3-mediated tau phosphorylation was significantly lower in adult bird brains than in mouse brains, a phenomenon that was reproduced in GSK-3α knockout mouse brains. Tau phosphorylation was detected in brains from bird embryos suggesting that GSK-3 isozymes play distinct roles in tau phosphorylation during development. Birds are natural GSK-3α knockout organisms and may serve as a novel model to study the distinct functions of GSK-3 isozymes. Copyright © 2011 Federation of European Biochemical Societies. All rights reserved.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

PubMed

Harris, Kimberly A; Zhou, Zhiyuan; Peters, Michelle L; Wilkins, Sarah G; Breaker, Ronald R

2018-06-18

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans , OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA ( ∆ole ) or OapA ( ∆oapA ) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans .

Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells

PubMed Central

Viiri, Leena E.; Vihervaara, Terhi; Koistinen, Kaisa M.; Hilvo, Mika; Ekroos, Kim; Käkelä, Reijo; Aalto-Setälä, Katriina

2017-01-01

ABSTRACT Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model to primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a three-step protocol through the definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid-metabolism-related genes were performed during the entire differentiation process from iPSCs to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very long FA were produced in the cells at a later stage of differentiation. This work uncovers the previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid-associated genes. Together with biochemical, functional and gene expression measurements, the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLC differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring, for example, the lipid metabolism involved in the development of fatty liver disease or atherosclerosis. PMID:28733363

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

PubMed

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

2017-06-01

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

Acetylcholinesterases of blood-feeding flies and ticks.

PubMed

Temeyer, Kevin B; Tuckow, Alexander P; Brake, Danett K; Li, Andrew Y; Pérez de León, Adalberto A

2013-03-25

Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimer's disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to elucidate structure-activity relationships for AChE that are important for advances in targeted pest control, as well as potential applications for medicine and biosecurity. Published by Elsevier Ireland Ltd.

Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme

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

Zhang, Yang; Zhu, Xuling; Torelli, Andrew T

2010-08-30

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind amore » [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the C γ,Met-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.« less

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

PubMed Central

Varshavsky, Alexander

2012-01-01

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca2+ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca2+-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca2+ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca2+ alone or Ca2+ and its ionophore (Erickson et al., Science 1978;199:1219–1221; Harris, Pharmacol Biochem Behav 1979;10:527–534; Erickson et al., Pharmacol Biochem Behav 1980;12:651–656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep. PMID:22930402

Structural diversity of supercoiled DNA

PubMed Central

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-01-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function. PMID:26455586

Structural diversity of supercoiled DNA

NASA Astrophysics Data System (ADS)

Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

2015-10-01

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

Botulinum neurotoxin homologs in non-Clostridium species.

PubMed

Mansfield, Michael J; Adams, Jeremy B; Doxey, Andrew C

2015-01-30

Clostridial neurotoxins (CNTs) are the deadliest toxins known and the causative agents of botulism and tetanus. Despite their structural and functional complexity, no CNT homologs are currently known outside Clostridium. Here, we report the first homologs of Clostridium CNTs within the genome of the rice fermentation organism Weissella oryzae SG25. One gene in W. oryzae S25 encodes a protein with a four-domain architecture and HExxH protease motif common to botulinum neurotoxins (BoNTs). An adjacent gene with partial similarity to CNTs is also present, and both genes seem to have been laterally transferred into the W. oryzae genome from an unknown source. Identification of mobile, CNT-related genes outside of Clostridium has implications for our understanding of the evolution of this important toxin family. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes

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

Ortega, Corrie; Frando, Andrew; Webb-Robertson, Bobbie-Jo

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of disease-causing Plasmodium falciparum (Pf) blood stage infection but also blocking parasite transmission from humans to mosquito to prevent disease spread. Numerous antimalarial drugs have become ineffective due to parasite drug resistance and many currently used therapies do not kill gametocytes, highly specialized sexual parasite stages with distinct physiology that are necessary for transmission from the human host to the mosquito vector. Further confounding next generation drug development against Pf is the lack of known biochemical activity for most parasite gene products as well as themore » unknown metabolic needs of non-replicating gametocyte. Here, we take a systematic activity-based proteomics approach to survey the large and druggable ATPase family that is associated with replicating blood stage asexual parasites and transmissible gametocytes. We experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. ATPase activity broadly changes during the transition from asexual schizonts to gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.« less

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

NASA Astrophysics Data System (ADS)

Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

2017-01-01

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

Microdissection of black widow spider silk-producing glands.

PubMed

Jeffery, Felicia; La Mattina, Coby; Tuton-Blasingame, Tiffany; Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-11

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

BioCluster: tool for identification and clustering of Enterobacteriaceae based on biochemical data.

PubMed

Abdullah, Ahmed; Sabbir Alam, S M; Sultana, Munawar; Hossain, M Anwar

2015-06-01

Presumptive identification of different Enterobacteriaceae species is routinely achieved based on biochemical properties. Traditional practice includes manual comparison of each biochemical property of the unknown sample with known reference samples and inference of its identity based on the maximum similarity pattern with the known samples. This process is labor-intensive, time-consuming, error-prone, and subjective. Therefore, automation of sorting and similarity in calculation would be advantageous. Here we present a MATLAB-based graphical user interface (GUI) tool named BioCluster. This tool was designed for automated clustering and identification of Enterobacteriaceae based on biochemical test results. In this tool, we used two types of algorithms, i.e., traditional hierarchical clustering (HC) and the Improved Hierarchical Clustering (IHC), a modified algorithm that was developed specifically for the clustering and identification of Enterobacteriaceae species. IHC takes into account the variability in result of 1-47 biochemical tests within this Enterobacteriaceae family. This tool also provides different options to optimize the clustering in a user-friendly way. Using computer-generated synthetic data and some real data, we have demonstrated that BioCluster has high accuracy in clustering and identifying enterobacterial species based on biochemical test data. This tool can be freely downloaded at http://microbialgen.du.ac.bd/biocluster/. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Structure-based functional annotation: yeast ymr099c codes for a D-hexose-6-phosphate mutarotase.

PubMed

Graille, Marc; Baltaze, Jean-Pierre; Leulliot, Nicolas; Liger, Dominique; Quevillon-Cheruel, Sophie; van Tilbeurgh, Herman

2006-10-06

Despite the generation of a large amount of sequence information over the last decade, more than 40% of well characterized enzymatic functions still lack associated protein sequences. Assigning protein sequences to documented biochemical functions is an interesting challenge. We illustrate here that structural genomics may be a reasonable approach in addressing these questions. We present the crystal structure of the Saccharomyces cerevisiae YMR099cp, a protein of unknown function. YMR099cp adopts the same fold as galactose mutarotase and shares the same catalytic machinery necessary for the interconversion of the alpha and beta anomers of galactose. The structure revealed the presence in the active site of a sulfate ion attached by an arginine clamp made by the side chain from two strictly conserved arginine residues. This sulfate is ideally positioned to mimic the phosphate group of hexose 6-phosphate. We have subsequently successfully demonstrated that YMR099cp is a hexose-6-phosphate mutarotase with broad substrate specificity. We solved high resolution structures of some substrate enzyme complexes, further confirming our functional hypothesis. The metabolic role of a hexose-6-phosphate mutarotase is discussed. This work illustrates that structural information has been crucial to assign YMR099cp to the orphan EC activity: hexose-phosphate mutarotase.

Phylogenetic profiles reveal structural/functional determinants of TRPC3 signal-sensing antennae

PubMed Central

Ko, Kyung Dae; Bhardwaj, Gaurav; Hong, Yoojin; Chang, Gue Su; Kiselyov, Kirill

2009-01-01

Biochemical assessment of channel structure/function is incredibly challenging. Developing computational tools that provide these data would enable translational research, accelerating mechanistic experimentation for the bench scientist studying ion channels. Starting with the premise that protein sequence encodes information about structure, function and evolution (SF&E), we developed a unified framework for inferring SF&E from sequence information using a knowledge-based approach. The Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST) provides phylogenetic profiles that can model, ab initio, SF&E relationships of biological sequences at the whole protein, single domain and single-amino acid level.1,2 In our recent paper,4 we have applied GDDA-BLAST analysis to study canonical TRP (TRPC) channels1 and empirically validated predicted lipid-binding and trafficking activities contained within the TRPC3 TRP_2 domain of unknown function. Overall, our in silico, in vitro, and in vivo experiments support a model in which TRPC3 has signal-sensing antennae which are adorned with lipid-binding, trafficking and calmodulin regulatory domains. In this Addendum, we correlate our functional domain analysis with the cryo-EM structure of TRPC3.3 In addition, we synthesize recent studies with our new findings to provide a refined model on the mechanism(s) of TRPC3 activation/deactivation. PMID:19704910

Expanding pH screening space using multiple droplets with secondary buffers for protein crystallization

NASA Astrophysics Data System (ADS)

Zhang, Chen-Yan; Dong, Chen; Lu, Xiao-Li; Wang, Bei; He, Tian-Yuan; Yang, Rui-Zeng; Lin, Hua-Long; Yang, Xue-Zhou; Yin, Da-Chuan

2017-04-01

We have proposed a rational strategy for selecting a suitable pH of protein solution based on protein biochemical properties. However, it is difficult to use this strategy for biochemical properties unknown proteins. In this paper, a simpler and faster pH buffer strategy was proposed. An additional pH-controlling buffer was added to crystallization droplet mixed with protein solution and commercial crystallization reagents to adjust its pH. The results revealed that protein crystallization success rates were enhanced by this strategy due to expansion of the pH screening space, which was closely related with protein solubility. Thus, the possibility of reaching supersaturation was increased by using this strategy.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: Insights into molecular recognition of invading pathogens in Manduca sexta.

PubMed

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R; Jiang, Haobo

2015-07-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. Copyright © 2015 Elsevier Ltd. All rights reserved.

EML proteins in microtubule regulation and human disease.

PubMed

Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

2016-10-15

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Phylogenetic analysis and expression profiling of the pattern recognition receptors: insights into molecular recognition of invading pathogens in Manduca sexta

PubMed Central

Zhang, Xiufeng; He, Yan; Cao, Xiaolong; Gunaratna, Ramesh T.; Chen, Yun-ru; Blissard, Gary; Kanost, Michael R.; Jiang, Haobo

2015-01-01

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect. PMID:25701384

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.

Neurocognitive Function in Acromegaly after Surgical Resection of GH-Secreting Adenoma versus Naïve Acromegaly

PubMed Central

Martín-Rodríguez, Juan Francisco; Madrazo-Atutxa, Ainara; Venegas-Moreno, Eva; Benito-López, Pedro; Gálvez, María Ángeles; Cano, David A.; Tinahones, Francisco J.; Torres-Vela, Elena; Soto-Moreno, Alfonso; Leal-Cerro, Alfonso

2013-01-01

Patients with active untreated acromegaly show mild to moderate neurocognitive disorders that are associated to chronic exposure to growth hormone (GH) and insulin-like growth factor (IGF-I) hypersecretion. However, it is unknown whether these disorders improve after controlling GH/IGF-I hypersecretion. The aim of this study was to compare neurocognitive functions of patients who successfully underwent GH-secreting adenoma transsphenoidal surgery (cured patients) with patients with naive acromegaly. In addition, we wanted to determine the impact of different clinical and biochemical variables on neurocognitive status in patients with active disease and after long-term cure. A battery of six standardized neuropsychological tests assessed attention, memory and executive functioning. In addition, a quantitative electroencephalography with Low-Resolution Electromagnetic Tomography (LORETA) solution was performed to obtain information about the neurophysiological state of the patients. Neurocognitive data was compared to that of a healthy control group. Multiple linear regression analysis was also conducted using clinical and hormonal parameters to obtain a set of independent predictors of neurocognitive state before and after cure. Both groups of patients scored significantly poorer than the healthy controls on memory tests, especially those assessing visual and verbal recall. Patients with cured acromegaly did not obtain better cognitive measures than naïve patients. Furthermore memory deficits were associated with decreased beta activity in left medial temporal cortex in both groups of patients. Regression analysis showed longer duration of untreated acromegaly was associated with more severe neurocognitive complications, regardless of the diagnostic group, whereas GH levels at the time of assessment was related to neurocognitive outcome only in naïve patients. Longer duration of post-operative biochemical remission of acromegaly was associated with better neurocognitive state. Overall, this data suggests that the effects of chronic exposure to GH/IGF-I hypersecretion could have long-term effects on brain functions. PMID:23593161

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

PubMed

Haid, A; Schweyen, R J; Bechmann, H; Kaudewitz, F; Solioz, M; Schatz, G

1979-03-01

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).

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

Of Mice, Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass

PubMed Central

Malda, Jos; de Grauw, Janny C.; Benders, Kim E. M.; Kik, Marja J. L.; van de Lest, Chris H. A.; Creemers, Laura B.; Dhert, Wouter J. A.; van Weeren, P. René

2013-01-01

Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue’s thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse) to 4000 kg (African elephant). Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content) and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content) decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications. PMID:23437402

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos*

PubMed Central

Hansen, Rasmus; Sønderkær, Mads; Arinell, Karin; Swenson, Jon E.; Revsbech, Inge G.

2016-01-01

Brown bears (Ursus arctos) hibernate for 5–7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general. PMID:27609515

Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial y-Glutamylcysteine Synthase

Treesearch

Chuanxin Ma; Sudesh Chhikara; Rakesh Minocha; Stephanie Long; Craig Musante; Jason C. White; Baoshan Xing; Om Parkash Dhankher

2015-01-01

Silver nanoparticles (Ag NPs) are widely used in consumer products, and their release has raised serious concerns about the risk of their exposure to the environment and to human health. However, biochemical mechanisms by which plants counteract NP toxicity are largely unknown. We have previously engineered Crambe abyssinica plants expressing the...

Serum Glutamic-Oxaloacetic Transaminase (GOT) and Glutamic-Pyruvic Transaminase (GPT) Levels in Children and Adolescents with Intellectual Disabilities

ERIC Educational Resources Information Center

Lin, Jin-Ding; Lin, Pei-Ying; Chen, Li-Mei; Fang, Wen-Hui; Lin, Lan-Ping; Loh, Ching-Hui

2010-01-01

The elevated serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) rate among people with intellectual disabilities (ID) is unknown and have not been sufficiently studies. The present paper aims to provide the profile of GOT and GPT, and their associated relationship with other biochemical levels of children or…

Biochemical basis for activation of virulence genes by bile salts in Vibrio parahaemolyticus.

PubMed

Rivera-Cancel, Giomar; Orth, Kim

2017-07-04

Bile salts act as a stressor to bacteria that transit the intestinal tract. Enteric pathogens have hijacked bile as an intestinal signal to regulate virulence factors. We recently demonstrated that Vibrio parahemolyticus senses bile salts via a heterodimeric receptor formed by the periplasmic domains of inner-membrane proteins VtrA and VtrC. Crystal structures of the periplasmic complex reveal that VtrA and VtrC form a β-barrel that binds bile salts in its hydrophobic interior to activate the VtrA cytoplasmic DNA-binding domain. Proteins with the same domain arrangement as VtrA and VtrC are widespread in Vibrio and related bacteria, where they are involved in regulating virulence and other unknown functions. Here we discuss our findings and review current knowledge on VtrA and VtrC homologs. We propose that signaling by these membrane-bound transcription factors can be advantageous for the regulation of membrane and secretory proteins.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

PubMed

Snyder, P M; Cheng, C; Prince, L S; Rogers, J C; Welsh, M J

1998-01-09

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

Single-platelet nanomechanics measured by high-throughput cytometry

NASA Astrophysics Data System (ADS)

Myers, David R.; Qiu, Yongzhi; Fay, Meredith E.; Tennenbaum, Michael; Chester, Daniel; Cuadrado, Jonas; Sakurai, Yumiko; Baek, Jong; Tran, Reginald; Ciciliano, Jordan C.; Ahn, Byungwook; Mannino, Robert G.; Bunting, Silvia T.; Bennett, Carolyn; Briones, Michael; Fernandez-Nieves, Alberto; Smith, Michael L.; Brown, Ashley C.; Sulchek, Todd; Lam, Wilbur A.

2017-02-01

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

Crystal structure of yeast allantoicase reveals a repeated jelly roll motif.

PubMed

Leulliot, Nicolas; Quevillon-Cheruel, Sophie; Sorel, Isabelle; Graille, Marc; Meyer, Philippe; Liger, Dominique; Blondeau, Karine; Janin, Joël; van Tilbeurgh, Herman

2004-05-28

Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea. The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown. In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 A by single anomalous diffraction. This constitutes the first structure for an enzyme of this pathway. The structure reveals a repeated jelly roll beta-sheet motif, also present in proteins of unrelated biochemical function. Allantoicase has a hexameric arrangement in the crystal (dimer of trimers). Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif. The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site.

Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.

PubMed

Brockly, Frédérique; Piechaczyk, Marc; Bossis, Guillaume

2016-01-01

SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

PubMed

Banadyga, Logan; Hoenen, Thomas; Ambroggio, Xavier; Dunham, Eric; Groseth, Allison; Ebihara, Hideki

2017-08-09

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis.

PubMed

Peng, Yuancheng; Chen, Liangliang; Li, Shengjun; Zhang, Yueying; Xu, Ran; Liu, Zupei; Liu, Wuxia; Kong, Jingjing; Huang, Xiahe; Wang, Yingchun; Cheng, Beijiu; Zheng, Leiying; Li, Yunhai

2018-04-18

Sugars function as signal molecules to regulate growth, development, and gene expression in plants, yeasts, and animals. A coordination of sugar availability with phytohormone signals is crucial for plant growth and development. The molecular link between sugar availability and hormone-dependent plant growth are largely unknown. Here we report that BRI1 and BAK1 are involved in sugar-responsive growth and development. Glucose influences the physical interactions and phosphorylations of BRI1 and BAK1 in a concentration-dependent manner. BRI1 and BAK1 physically interact with G proteins that are essential for mediating sugar signaling. Biochemical data show that BRI1 can phosphorylate G protein β subunit and γ subunits, and BAK1 can phosphorylate G protein γ subunits. Genetic analyses suggest that BRI1 and BAK1 function in a common pathway with G-protein subunits to regulate sugar responses. Thus, our findings reveal an important genetic and molecular mechanism by which BR receptors associate with G proteins to regulate sugar-responsive growth and development.

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

PubMed

Reviakine, Ilya

2018-02-12

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted. Copyright © 2018 Elsevier B.V. All rights reserved.

Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

PubMed Central

Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

2004-01-01

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

SIRT3 mediates multi-tissue coupling for metabolic fuel switching.

PubMed

Dittenhafer-Reed, Kristin E; Richards, Alicia L; Fan, Jing; Smallegan, Michael J; Fotuhi Siahpirani, Alireza; Kemmerer, Zachary A; Prolla, Tomas A; Roy, Sushmita; Coon, Joshua J; Denu, John M

2015-04-07

SIRT3 is a member of the Sirtuin family of NAD(+)-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations; however, the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild-type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis. Copyright © 2015 Elsevier Inc. All rights reserved.

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

NASA Astrophysics Data System (ADS)

Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

2016-08-01

Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2~Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

Peptidomics of prolyl endopeptidase in the central nervous system

PubMed Central

Nolte, Whitney M.; Tagore, Debarati M.; Lane, William S.; Saghatelian, Alan

2009-01-01

Prolyl endopeptidase (Prep) is a member of the prolyl peptidase family and is of interest due to its unique biochemistry and connections to cognitive function. Using an unbiased mass spectrometry (MS)-based peptidomics platform, we identified Prep regulated peptides in the central nervous system (CNS) of mice by measuring changes in the peptidome as a function of Prep activity. This approach was validated by the identification of known Prep substrates, such as the neuropeptide substance P and thymosin-β4, the precursor to the bioactive peptide Ac-SDKP. In addition to these known substrates, we also discovered that Prep regulates many additional peptides, including additional bioactive peptides and proline rich peptides (PRPs). Biochemical experiments confirmed that some of these Prep regulated peptides are indeed substrates of the enzyme. Moreover, these experiments also supported the known preference of Prep for shorter peptides, while revealing a previously unknown cleavage site specificity of Prep when processing certain multi-proline containing peptides, including PRPs. The discovery of Prep regulated peptides implicates Prep in new biological pathways and provides insights into the biochemistry of this enzyme. PMID:19911840

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression

PubMed Central

Schneider, Maren; Hellerschmied, Doris; Schubert, Tobias; Amlacher, Stefan; Vinayachandran, Vinesh; Reja, Rohit; Pugh, B. Franklin; Clausen, Tim; Köhler, Alwin

2015-01-01

Summary Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery. PMID:26317468

Study of improving the quality of bread and wheat-aegilops hybrids with the biotechnological ways

NASA Astrophysics Data System (ADS)

Ganbarzada, Aygun; Hasanova, Sudaba

2016-08-01

The great need of the people to bread demands to increase high qualitative grain plants. At present time for solving these problem different methods of biochemistry, genetics and molecular biology are widely used in the process of selection. To investigate biochemical peculiarities of wheat-aegilops hybrids and to define the correlative relation between these characteristics. To investigate the technological peculiarities of wheat- aegilops hybrids and to define the relation between their main biochemical and technological characteristics. The conclusion of this investigation showed the followings- the wheat-aegilops hybrids according to their morphological and biochemical characteristics have approached to wheats. The electrophoretic spectres of the wheat- aegilops hybrids which have stable for their morphological characteristics are homogeny and heterogenic. Hereditarily some group protein components have passed to their tribes from their parents. But spontaneous hybridisation results in taking part the components of other unknown wheats in these electrophoretic spectres. There is a relation between the electrophoretic spectres and the indications of the grain quality.

The microbiology "unknown" misadventure.

PubMed

Boyer, B; DeBenedictis, K J; Master, R; Jones, R S

1998-06-01

A 19-year-old nursing student was hospitalized after several days of nausea, vomiting, diarrhea, and fevers. Salmonella paratyphi A was isolated from multiple blood cultures. Because this is an unlikely isolate in the United States, an investigation ensued. Two and a half weeks earlier, the student had been working on a microbiology laboratory exercise "unknown." Both the "unknown" organism and the patient's blood culture isolates were identified as S. paratyphi A, with the same biochemical reactions and antimicrobial susceptibility results. The patient's condition improved with antibiotic therapy, and she was discharged after 9 days in the hospital. Conclusions related to our investigation are as follows: (1) relatively virulent organisms were unnecessary to fulfill the laboratory objectives, (2) pipetting by mouth must never be allowed, (3) proper labeling of specimens is imperative, (4) instructors should have knowledge of laboratory safety regulations, and (5) it is the obligation of laboratory directors and administrators to provide a safe academic environment.

FAP finds FGF21 easy to digest.

PubMed

Gillum, Matthew P; Potthoff, Matthew J

2016-05-01

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates carbohydrate and lipid metabolism. In humans, circulating FGF21 is inactivated by proteolytic cleavage of its C-terminus, thereby preventing signalling through a receptor complex. The mechanism for this cleavage event and the factors contributing to the post-translational regulation of FGF21 activity has previously been unknown. In a recent issue of the Biochemical Journal, Zhen et al. have identified fibroblast activation protein (FAP) as the endopeptidase responsible for this site-specific cleavage of human FGF21 (hFGF21), and propose that inhibition of FAP may be a therapeutic strategy to increase endogenous levels of active FGF21. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Syndromology: an updated conceptual overview. III. Syndrome delineation.

PubMed

Cohen, M M

1989-10-01

In Part III, the process of syndrome delineation is unfolded and its significance discussed. Unknown genesis syndrome categories include provisionally unique pattern syndromes and recurrent pattern syndromes. As more information becomes available, it is often possible to establish a Mendelian, chromosomal, teratogenic, or biochemical basis for the disorder in question. It has been estimated that newly recognized syndromes are being described at the rate of one or more per week. The process of syndrome delineation is not an academic exercise but actually fosters good patient care. As an unknown syndrome becomes delineated, its phenotypic spectrum, its natural history, and its recurrence risk become known, allowing for better patient care and family counseling.

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

PubMed Central

Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H.; Mondragón, Alfonso

2013-01-01

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

PubMed Central

Uusimaa, Johanna; Evans, Julie; Smith, Conrad; Butterworth, Anna; Craig, Kate; Ashley, Neil; Liao, Chunyan; Carver, Janet; Diot, Alan; Macleod, Lorna; Hargreaves, Iain; Al-Hussaini, Abdulrahman; Faqeih, Eissa; Asery, Ali; Al Balwi, Mohammed; Eyaid, Wafaa; Al-Sunaid, Areej; Kelly, Deirdre; van Mourik, Indra; Ball, Sarah; Jarvis, Joanna; Mulay, Arundhati; Hadzic, Nedim; Samyn, Marianne; Baker, Alastair; Rahman, Shamima; Stewart, Helen; Morris, Andrew AM; Seller, Anneke; Fratter, Carl; Taylor, Robert W; Poulton, Joanna

2014-01-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts. PMID:23714749

Advanced Nanoporous Materials for Micro-Gravimetric Sensing to Trace-Level Bio/Chemical Molecules

PubMed Central

Xu, Pengcheng; Li, Xinxin; Yu, Haitao; Xu, Tiegang

2014-01-01

Functionalized nanoporous materials have been developed recently as bio/chemical sensing materials. Due to the huge specific surface of the nano-materials for molecular adsorption, high hopes have been placed on gravimetric detection with micro/nano resonant cantilevers for ultra-sensitive sensing of low-concentration bio/chemical substances. In order to enhance selectivity of the gravimetric resonant sensors to the target molecules, it is crucial to modify specific groups onto the pore-surface of the nano-materials. By loading the nanoporous sensing material onto the desired region of the mass-type transducers like resonant cantilevers, the micro-gravimetric bio/chemical sensors can be formed. Recently, such micro-gravimetric bio/chemical sensors have been successfully applied for rapid or on-the-spot detection of various bio/chemical molecules at the trace-concentration level. The applicable nanoporous sensing materials include mesoporous silica, zeolite, nanoporous graphene oxide (GO) and so on. This review article focuses on the recent achievements in design, preparation, functionalization and characterization of advanced nanoporous sensing materials for micro-gravimetric bio/chemical sensing. PMID:25313499

Function-based Biosensor for Hazardous Waste Toxin Detection

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

James J Hickman

There is a need for new types of toxicity sensors in the DOE and other agencies that are based on biological function as the toxins encountered during decontamination or waste remediation may be previously unknown or their effects subtle. Many times the contents of the environmental waste, especially the minor components, have not been fully identified and characterized. New sensors of this type could target unknown toxins that cause death as well as intermediate levels of toxicity that impair function or cause long term impairment that may eventually lead to death. The primary question posed in this grant was tomore » create an electronically coupled neuronal cellular circuit to be used as sensor elements for a hybrid non-biological/biological toxin sensor system. A sensor based on the electrical signals transmitted between two mammalian neurons would allow the marriage of advances in solid state electronics with a functioning biological system to develop a new type of biosensor. Sensors of this type would be a unique addition to the field of sensor technology but would also be complementary to existing sensor technology that depends on knowledge of what is to be detected beforehand. We integrated physics, electronics, surface chemistry, biotechnology, and fundamental neuroscience in the development of this biosensor. Methods were developed to create artificial surfaces that enabled the patterning of discrete cells, and networks of cells, in culture; the networks were then aligned with transducers. The transducers were designed to measure electromagnetic fields (EMF) at low field strength. We have achieved all of the primary goals of the project. We can now pattern neurons routinely in our labs as well as align them with transducers. We have also shown the signals between neurons can be modulated by different biochemicals. In addition, we have made another significant advance where we have repeated the patterning results with adult hippocampal cells. Finally, we demonstrated that patterned cardiac cells on microelectrode arrays could act as sensors as well.« less

21 CFR 315.5 - Evaluation of effectiveness.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 601.34 - Evaluation of effectiveness.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 315.5 - Evaluation of effectiveness.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 315.5 - Evaluation of effectiveness.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 601.34 - Evaluation of effectiveness.

Code of Federal Regulations, 2014 CFR

2014-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 601.34 - Evaluation of effectiveness.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 315.5 - Evaluation of effectiveness.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 601.34 - Evaluation of effectiveness.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 601.34 - Evaluation of effectiveness.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

21 CFR 315.5 - Evaluation of effectiveness.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., physiological, or biochemical assessment is established by demonstrating in a defined clinical setting reliable measurement of function(s) or physiological, biochemical, or molecular process(es). (3) The claim of disease... demonstrating in a defined clinical setting that the test is useful in diagnostic or therapeutic patient...

Microdissection of Black Widow Spider Silk-producing Glands

PubMed Central

Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-01

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709

Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules.

PubMed

Secca, T; Sciaccaluga, M; Marra, A; Barberini, L; Bicchierai, M C

2011-04-01

In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs). However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system. Copyright © 2011 Elsevier Ltd. All rights reserved.

A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase–dependent Rap1 activation slows storage-induced platelet deterioration

PubMed Central

Schubert, Peter; Thon, Jonathan N.; Walsh, Geraldine M.; Chen, Cindy H.I.; Moore, Edwin D.; Devine, Dana V.; Kast, Juergen

2015-01-01

BACKGROUND The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown. STUDY DESIGN AND METHODS A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development. RESULTS Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin αIIbβ3 activation and release of α-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation. CONCLUSION These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase–dependent Rap1 activation leads to integrin αIIbβ3 activation and PLT degranulation. PMID:19497060

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis.

PubMed

Varshavsky, Alexander

2012-11-01

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca²⁺ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca²⁺-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca²⁺ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca²⁺ alone or Ca²⁺ and its ionophore (Erickson et al., Science 1978;199:1219-1221; Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep. Copyright © 2012 The Protein Society.

Phenotype of osteosarcopenia in older individuals with a history of falling.

PubMed

Huo, Ya Ruth; Suriyaarachchi, Pushpa; Gomez, Fernando; Curcio, Carmen L; Boersma, Derek; Muir, Susan W; Montero-Odasso, Manuel; Gunawardene, Piumali; Demontiero, Oddom; Duque, Gustavo

2015-04-01

In older persons, the combination of osteopenia/osteoporosis and sarcopenia has been proposed as a subset of frailer individuals at higher risk of institutionalization, falls, and fractures. However, the particular clinical, biochemical, and functional characteristics of the osteosarcopenic (OS) patients remain unknown. In this study, we used a clinical definition of osteosarcopenia aiming to determine the clinical, functional, and biochemical features that are unique to these patients within a population of older people who fall. Cross-sectional study. Falls and Fractures Clinic, Nepean Hospital (Penrith, NSW, Australia). A total of 680 people (mean age = 79, 65% women) assessed between 2009 and 2013. Assessment included medical history, physical examination, bone densitometry and body composition by dual-energy X-ray absorptiometry, posturography, grip strength, gait parameters (GaitRITE), and blood tests for nutrition and secondary causes of sarcopenia and osteoporosis. Patients were divided into 4 groups: (1) osteopenic (BMD <-1.0 SD), (2) sarcopenic, (3) OS, and (4) nonsarcopenic/nonosteopenic. Difference between groups was assessed with 1-way ANOVA and χ(2) analysis. Multivariable linear regression evaluated the association between the groups and measures of physical function. Multivariable logistic regression evaluated risk factors for being in the OS group. Mean age of the OS patients was 80.4 ± 7.0 years. Our analyses showed that OS patients are older, mostly women, are at high risk for depression and malnutrition, have body mass index lower than 25, and showed a higher prevalence of peptic disease, inflammatory arthritis, maternal hip fracture, history of atraumatic fracture, and impaired mobility. We have reported a set of characteristics that are highly prevalent in OS patients. This study could be used to inform the design of future trials and to develop interventions to prevent institutionalization and poor outcomes in this particular set of high-risk patients. Copyright © 2015 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.

The Blood Testis Barrier and Male Sexual Dysfunction following Spinal Cord Injury

DTIC Science & Technology

2015-10-01

sensory and motor deficits . In addition to these well-described pathological outcomes, a majority of men will also experience greatly diminished...underlying this deficit has previously been unknown. We have explored the effects of spinal trauma on tissues that exhibit “barrier” properties, or...immune cell infiltration. The goal of this project is to: 1) further elaborate the early and long-term biochemical, molecular and structural deficits

Streptococcus ovuberis sp. nov., isolated from a subcutaneous abscess in the udder of a sheep.

PubMed

Zamora, Leydis; Pérez-Sancho, Marta; Fernández-Garayzábal, Jose Francisco; Orden, Jose Antonio; Domínguez-Bernal, Gustavo; de la Fuente, Ricardo; Domínguez, Lucas; Vela, Ana Isabel

2017-11-01

One unidentified, Gram-stain-positive, catalase-negative coccus-shaped organism was recovered from a subcutaneous abscess of the udder of a sheep and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolate was tentatively assigned to the genus Streptococcus, although the organism did not appear to match any recognized species. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus and showed that the nearest phylogenetic relatives of the unknown coccus corresponded to Streptococcus moroccensis and Streptococcus cameli (95.9 % 16S rRNA gene sequence similarity). The sodA sequence analysis showed less than 89.3 % sequence similarity with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from close relatives of the genus Streptococcusby using biochemical tests. A mass spectrometry profile was also obtained for the novel isolate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a representative of a novel species of the genus Streptococcus, Streptococcus ovuberis sp. nov. The type strain of Streptococcus ovuberissp. nov. is VB15-00779 T (=CECT 9179 T =CCUG 69612 T ).

Characterization of Actinomyces Isolates from Infected Root Canals of Teeth: Description of Actinomyces radicidentis sp. nov.

PubMed Central

Collins, Matthew D.; Hoyles, Lesley; Kalfas, Sotos; Sundquist, Goran; Monsen, Tor; Nikolaitchouk, Natalia; Falsen, Enevold

2000-01-01

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733. PMID:10970390

Systematic inference of functional phosphorylation events in yeast metabolism.

PubMed

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Malonylome Analysis Reveals the Involvement of Lysine Malonylation in Metabolism and Photosynthesis in Cyanobacteria.

PubMed

Ma, Yanyan; Yang, Mingkun; Lin, Xiaohuang; Liu, Xin; Huang, Hui; Ge, Feng

2017-05-05

As a recently validated reversible post translational modification, lysine malonylation regulates diverse cellular processes from bacteria to mammals, but its existence and function in photosynthetic organisms remain unknown. Cyanobacteria are the most ancient group of photosynthetic prokaryotes and contribute about 50% of the total primary production on Earth. Previously, we reported the lysine acetylome in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Here we performed the first proteomic survey of lysine malonylation in Synechocystis using highly accurate tandem mass spectrometry in combination with affinity purification. We identified 598 lysine malonylation sites on 339 proteins with high confidence in total. A bioinformatic analysis suggested that these malonylated proteins may play various functions and were distributed in diverse subcellular compartments. Among them, many malonylated proteins were involved in cellular metabolism. The functional significance of lysine malonylation in the metabolic enzyme activity of phosphoglycerate kinase (PGK) was determined by site-specific mutagenesis and biochemical studies. Interestingly, 27 proteins involved in photosynthesis were found to be malonylated for the first time, suggesting that lysine malonylation may be involved in photosynthesis. Thus our results provide the first lysine malonylome in a photosynthetic organism and suggest a previously unexplored role of lysine malonylation in the regulation of metabolic processes and photosynthesis in Synechocystis as well as in other photosynthetic organisms.

Redox-linked Conformational Dynamics in Apoptosis Inducing Factor

PubMed Central

Sevrioukova, Irina F.

2009-01-01

Apoptosis inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH2-NAD charge-transfer complexes (CTC) proposed to be functionally important. To get a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the x-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine and Phe309 rings, (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site, and (iii) an extensive hydrogen-bonding network involving His453, a key residue undergoing a conformational switch to directly interact and orient the nicotinamide in position optimal for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with the biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH. PMID:19447115

Defining functional DNA elements in the human genome

PubMed Central

Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

Brief Report: Biochemical Correlates of Clinical Impairment in High Functioning Autism and Asperger's Disorder

ERIC Educational Resources Information Center

Kleinhans, Natalia M.; Richards, Todd; Weaver, Kurt E.; Liang, Olivia; Dawson, Geraldine; Aylward, Elizabeth

2009-01-01

Amygdala dysfunction has been proposed as a critical contributor to social impairment in autism spectrum disorders (ASD). The current study investigated biochemical abnormalities in the amygdala in 20 high functioning adults with autistic disorder or Asperger's disorder and 19 typically developing adults matched on age and IQ. Magnetic resonance…

Predictive value of plasma β2-microglobulin on human body function and senescence.

PubMed

Dong, X-M; Cai, R; Yang, F; Zhang, Y-Y; Wang, X-G; Fu, S-L; Zhang, J-R

2016-06-01

To explore the correlation between plasma β2-microglobulin (β2-MG) as senescence factor with age, heart, liver and kidney function as well as the predictive value of β2-MG in human metabolism function and senescence. 387 cases of healthy people of different ages were selected and the automatic biochemical analyzer was used to test β2-MG in plasma based on immunoturbidimetry and also all biochemical indexes. The correlation between β2-MG and age, gender and all biochemical indexes was analyzed. β2-MG was positively correlated to age, r = 0.373; and the difference was of statistical significance (p < 0.010). It was significantly negative correlated to HDL-C but positively correlated to LP (a), BUN, CREA, UA, CYS-C, LDH, CK-MB, HBDH, AST, GLB and HCY. β2-MG was closely correlated to age, heart, kidney and liver biochemical indexes, which can be taken as an important biomarker for human body function and anti-senescence and have significant basic research and clinical guidance values.

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

Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.

PubMed

Neuwald, Andrew F; Altschul, Stephen F

2016-12-01

Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).

Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis*

PubMed Central

Shchedrina, Valentina A.; Everley, Robert A.; Zhang, Yan; Gygi, Steven P.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Selenoprotein K (SelK) is an 11-kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, selenoprotein S (SelS), and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins, and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had higher affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element, and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, and STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the oligosaccharyltransferase complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes. PMID:22016385

Biochemical and Biophysical Cues in Matrix Design for Chronic and Diabetic Wound Treatment

PubMed Central

Xiao, Yun; Ahadian, Samad

2017-01-01

Progress in biomaterial science and engineering and increasing knowledge in cell biology have enabled us to develop functional biomaterials providing appropriate biochemical and biophysical cues for tissue regeneration applications. Tissue regeneration is particularly important to treat chronic wounds of people with diabetes. Understanding and controlling the cellular microenvironment of the wound tissue are important to improve the wound healing process. In this study, we review different biochemical (e.g., growth factors, peptides, DNA, and RNA) and biophysical (e.g., topographical guidance, pressure, electrical stimulation, and pulsed electromagnetic field) cues providing a functional and instructive acellular matrix to heal diabetic chronic wounds. The biochemical and biophysical signals generally regulate cell–matrix interactions and cell behavior and function inducing the tissue regeneration for chronic wounds. Some technologies and devices have already been developed and used in the clinic employing biochemical and biophysical cues for wound healing applications. These technologies can be integrated with smart biomaterials to deliver therapeutic agents to the wound tissue in a precise and controllable manner. This review provides useful guidance in understanding molecular mechanisms and signals in the healing of diabetic chronic wounds and in designing instructive biomaterials to treat them. PMID:27405960

Odontometric sex estimation from clinically extracted molar teeth in a North Indian population sample.

PubMed

Tabasum, Qutsia; Sehrawat, Jagmahender Singh; Talwar, Manjit Kaur; Pathak, Raj Kamal

2017-01-01

Different dental features have contributed significantly toward sex determination in the forensic anthropological contexts. Population-specific standards (discriminant functions or regression formulae) have been suggested for various population groups to identify the sex of an unknown individual from dental dimensions and other odontometric features. The main purpose of the present investigation was to examine the degree of sexual dimorphism exhibited by the human teeth of North Indians and identify importance as a forensic tool in sex determination. The linear and diagonal dimensions were recorded at both crown and cementoenamel junction levels of 58 upper and 72 lower molars of 130 Northwest Indian subjects (73 males and 57 females). The measurements were subjected to appropriate statistical analyses to estimate the sex estimation accuracy from lower and upper molars separately. Univariate analyses revealed that molar teeth had greater dimensions in males than the females and the mesiodistal cervical diameter (MDCV) was found to be the most suitable variable for sex determination of the molars. The classification results were in agreement with the previously conducted studies. The index of sexual dimorphism (ISD) was calculated to be higher in lower molars than the upper molars, and the highest sex differences were observed for MDCV based on the ISD. The overall sex estimation accuracy obtained from multivariate discriminant function analysis and regression analysis of pooled data was 70.0% (74% males, 64.9% females) and 66.9% (78.1% males, 52.6% females), respectively. Odontometrics can play a significant role in establishing the biological identity of an unknown individual even from a single tooth in the absence of other sophisticated molecular or biochemical techniques used for this purpose.

Odontometric sex estimation from clinically extracted molar teeth in a North Indian population sample

PubMed Central

Tabasum, Qutsia; Sehrawat, Jagmahender Singh; Talwar, Manjit Kaur; Pathak, Raj Kamal

2017-01-01

Background: Different dental features have contributed significantly toward sex determination in the forensic anthropological contexts. Population-specific standards (discriminant functions or regression formulae) have been suggested for various population groups to identify the sex of an unknown individual from dental dimensions and other odontometric features. The main purpose of the present investigation was to examine the degree of sexual dimorphism exhibited by the human teeth of North Indians and identify importance as a forensic tool in sex determination. Materials and Methods: The linear and diagonal dimensions were recorded at both crown and cementoenamel junction levels of 58 upper and 72 lower molars of 130 Northwest Indian subjects (73 males and 57 females). The measurements were subjected to appropriate statistical analyses to estimate the sex estimation accuracy from lower and upper molars separately. Results: Univariate analyses revealed that molar teeth had greater dimensions in males than the females and the mesiodistal cervical diameter (MDCV) was found to be the most suitable variable for sex determination of the molars. The classification results were in agreement with the previously conducted studies. The index of sexual dimorphism (ISD) was calculated to be higher in lower molars than the upper molars, and the highest sex differences were observed for MDCV based on the ISD. The overall sex estimation accuracy obtained from multivariate discriminant function analysis and regression analysis of pooled data was 70.0% (74% males, 64.9% females) and 66.9% (78.1% males, 52.6% females), respectively. Conclusions: Odontometrics can play a significant role in establishing the biological identity of an unknown individual even from a single tooth in the absence of other sophisticated molecular or biochemical techniques used for this purpose. PMID:29657498

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.

Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction Support Anthrahydroquinone Reduction as the Physiological Function of Fungal 17β-Hydroxysteroid Dehydrogenase.

PubMed

Fürtges, Leon; Conradt, David; Schätzle, Michael A; Singh, Shailesh Kumar; Kraševec, Nada; Rižner, Tea Lanišnik; Müller, Michael; Husain, Syed Masood

2017-01-03

17β-Hydroxysteroid dehydrogenase (17β-HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)-dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17β-HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17β-HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17β-HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17β-HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17β-HSDcl in the biosynthesis of other anthrahydroquinone-derived natural products is proposed; hence, 17β-HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lipid transfer protein 3 as a target of MYB96 mediates freezing and drought stress in Arabidopsis

PubMed Central

Yang, Shuhua

2013-01-01

Several lipid-transfer proteins were reported to modulate the plant response to biotic stress; however, whether lipid-transfer proteins are also involved in abiotic stress remains unknown. This study characterized the function of a lipid-transfer protein, LTP3, during freezing and drought stress. LTP3 was expressed ubiquitously and the LTP3 protein was localized to the cytoplasm. A biochemical study showed that LTP3 was able to bind to lipids. Overexpression of LTP3 resulted in constitutively enhanced freezing tolerance without affecting the expression of CBFs and their target COR genes. Further analyses showed that LTP3 was positively regulated by MYB96 via the direct binding to the LTP3 promoter; consistently, transgenic plants overexpressing MYB96 exhibited enhanced freezing tolerance. This study also found that the loss-of-function mutant ltp3 was sensitive to drought stress, whereas overexpressing plants were drought tolerant, phenotypes reminiscent of myb96 mutant plants and MYB96-overexpressing plants. Taken together, these results demonstrate that LTP3 acts as a target of MYB96 to be involved in plant tolerance to freezing and drought stress. PMID:23404903

Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: close structural resemblance to E. coli pantothenate kinase.

PubMed

Li de La Sierra-Gallay, Ines; Collinet, Bruno; Graille, Marc; Quevillon-Cheruel, Sophie; Liger, Dominique; Minard, Philippe; Blondeau, Karine; Henckes, Gilles; Aufrère, Robert; Leulliot, Nicolas; Zhou, Cong-Zhao; Sorel, Isabelle; Ferrer, Jean-Luc; Poupon, Anne; Janin, Joël; van Tilbeurgh, Herman

2004-03-01

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of beta-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Copyright 2004 Wiley-Liss, Inc.

Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors*

PubMed Central

Paczkowski, Jon E.; Mukherjee, Sampriti; McCready, Amelia R.; Cong, Jian-Ping; Aquino, Christopher J.; Kim, Hahn; Henke, Brad R.; Smith, Chari D.; Bassler, Bonnie L.

2017-01-01

Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms. PMID:28119451

WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes.

PubMed

Jia, Da; Gomez, Timothy S; Metlagel, Zoltan; Umetani, Junko; Otwinowski, Zbyszek; Rosen, Michael K; Billadeau, Daniel D

2010-06-08

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

GmCYP82A3, a Soybean Cytochrome P450 Family Gene Involved in the Jasmonic Acid and Ethylene Signaling Pathway, Enhances Plant Resistance to Biotic and Abiotic Stresses

PubMed Central

Yan, Qiang; Cui, Xiaoxia; Lin, Shuai; Gan, Shuping; Xing, Han; Dou, Daolong

2016-01-01

The cytochrome P450 monooxygenases (P450s) represent a large and important enzyme superfamily in plants. They catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways, P450s are involved in a variety of metabolic pathways and participate in the homeostasis of phytohormones. The CYP82 family genes specifically reside in dicots and are usually induced by distinct environmental stresses. However, their functions are largely unknown, especially in soybean (Glycine max L.). Here, we report the function of GmCYP82A3, a gene from soybean CYP82 family. Its expression was induced by Phytophthora sojae infection, salinity and drought stresses, and treatment with methyl jasmonate (MeJA) or ethephon (ETH). Its expression levels were consistently high in resistant cultivars. Transgenic Nicotiana benthamiana plants overexpressing GmCYP82A3 exhibited strong resistance to Botrytis cinerea and Phytophthora parasitica, and enhanced tolerance to salinity and drought stresses. Furthermore, transgenic plants were less sensitive to jasmonic acid (JA), and the enhanced resistance was accompanied with increased expression of the JA/ET signaling pathway-related genes. PMID:27588421

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

PubMed

Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

2018-04-23

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

A new metal binding domain involved in cadmium, cobalt and zinc transport

DOE PAGES

Smith, Aaron T.; Barupala, Dulmini; Stemmler, Timothy L.; ...

2015-07-20

The P 1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P 1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. In this paper, we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P 1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd 2+, Co 2+ or Zn 2+ ions in distinctmore » and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Finally, taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P 1B-ATPases.« less

Void space inside the developing seed of Brassica napus and the modelling of its function

PubMed Central

Verboven, Pieter; Herremans, Els; Borisjuk, Ljudmilla; Helfen, Lukas; Ho, Quang Tri; Tschiersch, Henning; Fuchs, Johannes; Nicolaï, Bart M; Rolletschek, Hardy

2013-01-01

The developing seed essentially relies on external oxygen to fuel aerobic respiration, but it is currently unknown how oxygen diffuses into and within the seed, which structural pathways are used and what finally limits gas exchange. By applying synchrotron X-ray computed tomography to developing oilseed rape seeds we uncovered void spaces, and analysed their three-dimensional assembly. Both the testa and the hypocotyl are well endowed with void space, but in the cotyledons, spaces were small and poorly inter-connected. In silico modelling revealed a three orders of magnitude range in oxygen diffusivity from tissue to tissue, and identified major barriers to gas exchange. The oxygen pool stored in the voids is consumed about once per minute. The function of the void space was related to the tissue-specific distribution of storage oils, storage protein and starch, as well as oxygen, water, sugars, amino acids and the level of respiratory activity, analysed using a combination of magnetic resonance imaging, specific oxygen sensors, laser micro-dissection, biochemical and histological methods. We conclude that the size and inter-connectivity of void spaces are major determinants of gas exchange potential, and locally affect the respiratory activity of a developing seed. PMID:23692271

An effector Peptide family required for Drosophila toll-mediated immunity.

PubMed

Clemmons, Alexa W; Lindsay, Scott A; Wasserman, Steven A

2015-04-01

In Drosophila melanogaster, recognition of an invading pathogen activates the Toll or Imd signaling pathway, triggering robust upregulation of innate immune effectors. Although the mechanisms of pathogen recognition and signaling are now well understood, the functions of the immune-induced transcriptome and proteome remain much less well characterized. Through bioinformatic analysis of effector gene sequences, we have defined a family of twelve genes - the Bomanins (Boms) - that are specifically induced by Toll and that encode small, secreted peptides of unknown biochemical activity. Using targeted genome engineering, we have deleted ten of the twelve Bom genes. Remarkably, inactivating these ten genes decreases survival upon microbial infection to the same extent, and with the same specificity, as does eliminating Toll pathway function. Toll signaling, however, appears unaffected. Assaying bacterial load post-infection in wild-type and mutant flies, we provide evidence that the Boms are required for resistance to, rather than tolerance of, infection. In addition, by generating and assaying a deletion of a smaller subset of the Bom genes, we find that there is overlap in Bom activity toward particular pathogens. Together, these studies deepen our understanding of Toll-mediated immunity and provide a new in vivo model for exploration of the innate immune effector repertoire.

Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism

NASA Astrophysics Data System (ADS)

Torres, Idelisse Ortiz; Kuchenbecker, Kristopher M.; Nnadi, Chimno I.; Fletterick, Robert J.; Kelly, Mark J. S.; Fujimori, Danica Galonić

2015-02-01

The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin.

A Geometric Method for Model Reduction of Biochemical Networks with Polynomial Rate Functions.

PubMed

Samal, Satya Swarup; Grigoriev, Dima; Fröhlich, Holger; Weber, Andreas; Radulescu, Ovidiu

2015-12-01

Model reduction of biochemical networks relies on the knowledge of slow and fast variables. We provide a geometric method, based on the Newton polytope, to identify slow variables of a biochemical network with polynomial rate functions. The gist of the method is the notion of tropical equilibration that provides approximate descriptions of slow invariant manifolds. Compared to extant numerical algorithms such as the intrinsic low-dimensional manifold method, our approach is symbolic and utilizes orders of magnitude instead of precise values of the model parameters. Application of this method to a large collection of biochemical network models supports the idea that the number of dynamical variables in minimal models of cell physiology can be small, in spite of the large number of molecular regulatory actors.

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases

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

Bradshaw, Niels; Levdikov, Vladimir M.; Zimanyi, Christina M.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activitymore » in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.« less

Take time to smell the frogs: vocal sac glands of reed frogs (Anura: Hyperoliidae) contain species-specific chemical cocktails

PubMed Central

Starnberger, Iris; Poth, Dennis; Peram, Pardha Saradhi; Schulz, Stefan; Vences, Miguel; Knudsen, Jette; Barej, Michael F; Rödel, Mark-Oliver; Walzl, Manfred; Hödl, Walter

2013-01-01

Males of all reed frog species (Anura: Hyperoliidae) have a prominent, often colourful, gular patch on their vocal sac, which is particularly conspicuous once the vocal sac is inflated. Although the presence, shape, and form of the gular patch are well-known diagnostic characters for these frogs, its function remains unknown. By integrating biochemical and histological methods, we found strong evidence that the gular patch is a gland producing volatile compounds, which might be emitted while calling. Volatile compounds were confirmed by gas chromatography–mass spectrometry in the gular glands in 11 species of the hyperoliid genera Afrixalus, Heterixalus, Hyperolius, and Phlyctimantis. Comparing the gular gland contents of 17 specimens of four sympatric Hyperolius species yielded a large variety of 65 compounds in species-specific combinations. We suggest that reed frogs might use a complex combination of at least acoustic and chemical signals in species recognition and mate choice. PMID:24277973

Biochemical basis for activation of virulence genes by bile salts in Vibrio parahaemolyticus

PubMed Central

2017-01-01

ABSTRACT Bile salts act as a stressor to bacteria that transit the intestinal tract. Enteric pathogens have hijacked bile as an intestinal signal to regulate virulence factors. We recently demonstrated that Vibrio parahemolyticus senses bile salts via a heterodimeric receptor formed by the periplasmic domains of inner-membrane proteins VtrA and VtrC. Crystal structures of the periplasmic complex reveal that VtrA and VtrC form a β-barrel that binds bile salts in its hydrophobic interior to activate the VtrA cytoplasmic DNA-binding domain. Proteins with the same domain arrangement as VtrA and VtrC are widespread in Vibrio and related bacteria, where they are involved in regulating virulence and other unknown functions. Here we discuss our findings and review current knowledge on VtrA and VtrC homologs. We propose that signaling by these membrane-bound transcription factors can be advantageous for the regulation of membrane and secretory proteins. PMID:28129014

PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

NASA Astrophysics Data System (ADS)

de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

2010-08-01

Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

[Diagnosis and differential diagnosis of pancreatitis--diagnostic relevance of clinical and biochemical changes during the course of the disease and of endoscopic retrograde pancreatography (author's transl)].

PubMed

Ammann, R

1976-08-01

In order to classify a patient with pancreatitis according to the Marseille clasiffication the following criteria must be fulfilled: (a) an acute attach of pancreatitis must be observed, (b) the cause of pancreatitis has to be established, (c) the patient has to be followed over longer periods of time in order to find out, whether the process becomes chronic (involving progredient endocrine and exocrine insufficiency). Diagnostic problems of acute pancreatitis, relapsing pancreatitis, and chronic pancreatitis are discussed taking into account the author's own results. It is concluded, that repeated tests of pancreatitis function and demonstration of pancreatic calcification are more important for establishing the diagnosis of chronic pancreatitis than studies of pancreatic morphology including endoscopic retrograde pancreatography (ERP). ERP may help to find the cause of relapsing pancreatitis of unknown origine; it may help as well preoperatively to diagnose local changes of the pancreatic duct system in chronic pancreatitis.

Role of Fanconi Anemia FANCG in Preventing Double-Strand Breakage and Chromosomal Rearrangement during DNA Replication

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

Tebbs, R S; Hinz, J M; Yamada, N A

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are unknown. By constructing and characterizing a null fancg mutant of hamster CHO cells, we present several new insights for FA. The fancg cells show a broad sensitivity to genotoxic agents, not supporting the conventional concept of sensitivity to only DNA crosslinking agents. The aprt mutation rate is normal, but hprt mutations are reduced, which we ascribe to the lethality of large deletions. CAD and dhfr gene amplification rates are increased, implying excess chromosomal breakage during DNA replication, andmore » suggesting amplification as a contributing factor to cancer-proneness in FA patients. In S-phase cells, both spontaneous and mutagen-induced Rad51 nuclear foci are elevated. These results support a model in which FancG protein helps to prevent collapse of replication forks by allowing translesion synthesis or lesion bypass through homologous recombination.« less

Cdk5: one of the links between senile plaques and neurofibrillary tangles?

PubMed

Lee, Ming-Sum; Tsai, Li-Huei

2003-04-01

The relationship between amyloid plaques and neurofibrillary tangles, the two pathologic hallmarks of Alzheimer's disease (AD), is an unknown and controversial subject. However, emerging evidence from genetic and biochemical studies suggests that accumulation of amyloid beta peptides may play a causative role in AD pathogenesis. This led to the amyloid hypothesis, which proposes that amyloid beta peptides disrupt neuronal metabolic and ionic homeostasis and cause aberrant activation of kinases and/or inhibition of phosphatases. The resulting alteration in kinase and phosphatase activities ultimately leads to hyperphosphorylation of tau and formation of neurofibrillary tangles. Cyclin-dependent kinase 5 (Cdk5) is a tau kinase whose activity is induced by amyloid beta peptides. Its deregulation may represent one of the signal transduction pathways that connect amyloid beta toxicity to tau hyperphosphorylation. This article reviews the functions and regulation of Cdk5. Evidence that suggests deregulation of Cdk5 activity in AD by virtue of calpain cleavage of its activator p35 to p25 will be discussed.

SSD1, which encodes a plant-specific novel protein, controls plant elongation by regulating cell division in rice.

PubMed

Asano, Kenji; Miyao, Akio; Hirochika, Hirohiko; Kitano, Hidemi; Matsuoka, Makoto; Ashikari, Motoyuki

2010-01-01

Plant height is one of the most important traits in crop improvement. Therefore revealing the mechanism of plant elongation and controlling plant height in accordance with breeding object is important. In this study we analyzed a novel dwarf mutant, ssd1, of which phenotype is different from typical GA- or BR-related dwarf phenotype. ssd1 exhibits pleiotropic defects in elongation of various organs such as stems, roots, leaves, and flowers. ssd1 also shows abnormal cell files and shapes, which suggests defects of normal cell division in the mutant. Map-based cloning and complementation test demonstrated that the dwarf phenotype in ssd1 mutant was caused by insertion of retrotransposon in a gene, which encodes plant-specific protein with unknown biochemical function. A BLAST search revealed that SSD1-like genes exist in diverse plant species, including monocots and dicots, but not fern and moss. Our results demonstrate that SSD1 controls plant elongation by controlling cell division in higher plants.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

PubMed

Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

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

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

PubMed Central

Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

2014-01-01

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level. PMID:24639361

On the transmethylation hypothesis: stress, N,N-dimethyltryptamine, and positive symptoms of psychosis.

PubMed

Grammenos, Dionysios; Barker, Steven A

2015-06-01

Past research suggests a relationship between stress and positive symptoms of psychosis. However, the biological substrate of this relationship remains unknown. According to the transmethylation hypothesis, schizophrenia could result from a biochemical disruption in the stress mechanism. This biochemical disruption would lead to the production of a substance that would account for the symptoms of psychosis. Moreover, some studies have tested endogenous N,N-dimethyltryptamine (DMT) in the context of the transmethylation hypothesis. Stress has been found to elevate DMT levels in rodents. Also, elevated DMT levels have been associated with positive features of psychosis in psychiatric patients. Additionally, healthy participants treated with exogenous DMT experience predominantly positive symptoms of psychosis. The present paper examines endogenous DMT as a possible biological mediator of the relationship between stress and positive symptoms of psychosis.

Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

PubMed

Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

2013-03-01

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

PubMed

Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

2015-06-01

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

Physiologic and biochemical measurements and response to noxious stimulation at various concentrations of MS-222 in koi (Cyprinus carpio).

PubMed

Stockman, Jonathan; Weber, Ernest Scott P; Kass, Philip H; Pascoe, Peter J; Paul-Murphy, Joanne

2013-01-01

To evaluate the physiological effect and response to noxious stimulation at five concentrations of MS-222 in koi (Cyprinus carpio). Prospective experimental study. Twenty-one healthy adult unknown sex koi fish weighing mean 450±SD 120 g. Each fish was exposed to five different concentrations of MS-222 (50, 70, 110, 150 and 190 mg L(-1) ) in a random sequence during the same anaesthetic event. For each concentration of MS-222, vital functions such as heart rate (HR) (via Doppler) and opercular rate (OpR) were recorded after a standardized induction period. Response to two noxious stimuli in the form of haemostat clamp pressure applied on the tail and the lip was evaluated, and blood was drawn to measure biochemical and blood gas values. Decrease in response to noxious stimulation with an increase of MS-222 concentration both for the lip (p=0.0027) and the tail (p<0.0001) stimulus was observed. Biochemical values were unaffected by the concentration of MS-222 with the exception of lactate concentration which was weakly correlated with the duration of anaesthesia (r=0.31, p<0.001) and the number of times the fish was clamped or bled prior to sampling (r=0.23, p<0.001). Opercular rate decreased with the increase in anaesthetic concentration, and HR was not affected. Our results indicated a decrease in response to stimulus and a decrease in OpR that were associated with increased concentrations of MS-222. This may assist in establishing anaesthetic protocols using MS-222 in fish and supports the use of supramaximal pressure stimuli to teleost fish under variable MS-222 concentrations as a model for future studies. © 2012 The Authors. Veterinary Anaesthesia and Analgesia. © 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists.

Genetic and Biochemical Evidence That Haploinsufficiency of the Nf1 Tumor Suppressor Gene Modulates Melanocyte and Mast Cell Fates in Vivo

PubMed Central

Ingram, David A.; Yang, Feng-Chun; Travers, Jeffrey B.; Wenning, Mary Jo; Hiatt, Kelly; New, Sheryl; Hood, Antoinette; Shannon, Kevin; Williams, David A.; Clapp, D. Wade

2000-01-01

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's “two hit” model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1−/− murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W41 mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras–mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W41) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types. PMID:10620616

Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

PubMed

Ingram, D A; Yang, F C; Travers, J B; Wenning, M J; Hiatt, K; New, S; Hood, A; Shannon, K; Williams, D A; Clapp, D W

2000-01-03

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

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].

Does Oral Coenzyme Q10 Plus NADH Supplementation Improve Fatigue and Biochemical Parameters in Chronic Fatigue Syndrome?

PubMed Central

Cordero, Mario D.; Segundo, María José; Sáez-Francàs, Naia; Calvo, Natalia; Román-Malo, Lourdes; Aliste, Luisa; Fernández de Sevilla, Tomás; Alegre, José

2015-01-01

Abstract Chronic fatigue syndrome (CFS) is a chronic and extremely debilitating illness characterized by prolonged fatigue and multiple symptoms with unknown cause, diagnostic test, or universally effective treatment. Inflammation, oxidative stress, mitochondrial dysfunction, and CoQ10 deficiency have been well documented in CFS. We conducted an 8-week, randomized, double-blind placebo-controlled trial to evaluate the benefits of oral CoQ10 (200 mg/day) plus NADH (20 mg/day) supplementation on fatigue and biochemical parameters in 73 Spanish CFS patients. This study was registered in ClinicalTrials.gov (NCT02063126). A significant improvement of fatigue showing a reduction in fatigue impact scale total score (p<0.05) was reported in treated group versus placebo. In addition, a recovery of the biochemical parameters was also reported. NAD+/NADH (p<0.001), CoQ10 (p<0.05), ATP (p<0.05), and citrate synthase (p<0.05) were significantly higher, and lipoperoxides (p<0.05) were significantly lower in blood mononuclear cells of the treated group. These observations lead to the hypothesis that the oral CoQ10 plus NADH supplementation could confer potential therapeutic benefits on fatigue and biochemical parameters in CFS. Larger sample trials are warranted to confirm these findings. Antioxid. Redox Signal. 22, 679–685. PMID:25386668

Streptococcus pharyngis sp. nov., a novel streptococcal species isolated from the respiratory tract of wild rabbits.

PubMed

Vela, Ana I; Casas-Díaz, Encarna; Lavín, Santiago; Domínguez, Lucas; Fernández-Garayzábal, Jose F

2015-09-01

Four isolates of an unknown Gram-stain-positive, catalase-negative coccus-shaped organism, isolated from the pharynx of four wild rabbits, were characterized by phenotypic and molecular genetic methods. The micro-organisms were tentatively assigned to the genus Streptococcus based on cellular morphological and biochemical criteria, although the organisms did not appear to correspond to any species with a validly published name. Comparative 16S rRNA gene sequencing confirmed their identification as members of the genus Streptococcus, being most closely related phylogenetically to Streptococcus porcorum 682-03(T) (96.9% 16S rRNA gene sequence similarity). Analysis of rpoB and sodA gene sequences showed divergence values between the novel species and S. porcorum 682-03(T) (the closest phylogenetic relative determined from 16S rRNA gene sequences) of 18.1 and 23.9%, respectively. The novel bacterial isolate could be distinguished from the type strain of S. porcorum by several biochemical characteristics, such as the production of glycyl-tryptophan arylamidase and α-chymotrypsin, and the non-acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be assigned to a novel species of the genus Streptococcus, and named Streptococcus pharyngis sp. nov. The type strain is DICM10-00796B(T) ( = CECT 8754(T) = CCUG 66496(T)).

Biochemical tests of placental function for assessment in pregnancy.

PubMed

Neilson, James P

2012-08-15

Biochemical tests of placental or feto-placental function were widely used in the 1960s and 1970s in high-risk pregnancies to try to predict, and thus try to avoid, adverse fetal outcome. To assess the effects of performing biochemical tests of placental function in high-risk, low-risk, or unselected pregnancies. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (10 May 2012). Controlled trials (randomized or 'quasi-randomized') that compare the use of biochemical tests of placental function in pregnancy with non-use. Trial quality was assessed and data were extracted by the review author. A single eligible trial of poor quality was identified. It involved 622 women with high-risk pregnancies who had had plasma (o)estriol estimations. Women were allocated to have their (o)estriol results revealed or concealed on the basis of hospital record number (with attendant risk of selection bias). There were no obvious differences in perinatal mortality (relative risk (RR) 0.88, 95% confidence interval (CI) 0.36 to 2.13) or planned delivery (RR 0.97, 95% CI 0.81 to 1.15) between the two groups. The available trial data do not support the use of (o)estriol estimation in high-risk pregnancies. The single small trial available does not have the power to exclude a beneficial effect but this is probably of historical interest since biochemical testing has been superseded by biophysical testing in antepartum fetal assessment.

The Validity of Quasi-Steady-State Approximations in Discrete Stochastic Simulations

PubMed Central

Kim, Jae Kyoung; Josić, Krešimir; Bennett, Matthew R.

2014-01-01

In biochemical networks, reactions often occur on disparate timescales and can be characterized as either fast or slow. The quasi-steady-state approximation (QSSA) utilizes timescale separation to project models of biochemical networks onto lower-dimensional slow manifolds. As a result, fast elementary reactions are not modeled explicitly, and their effect is captured by nonelementary reaction-rate functions (e.g., Hill functions). The accuracy of the QSSA applied to deterministic systems depends on how well timescales are separated. Recently, it has been proposed to use the nonelementary rate functions obtained via the deterministic QSSA to define propensity functions in stochastic simulations of biochemical networks. In this approach, termed the stochastic QSSA, fast reactions that are part of nonelementary reactions are not simulated, greatly reducing computation time. However, it is unclear when the stochastic QSSA provides an accurate approximation of the original stochastic simulation. We show that, unlike the deterministic QSSA, the validity of the stochastic QSSA does not follow from timescale separation alone, but also depends on the sensitivity of the nonelementary reaction rate functions to changes in the slow species. The stochastic QSSA becomes more accurate when this sensitivity is small. Different types of QSSAs result in nonelementary functions with different sensitivities, and the total QSSA results in less sensitive functions than the standard or the prefactor QSSA. We prove that, as a result, the stochastic QSSA becomes more accurate when nonelementary reaction functions are obtained using the total QSSA. Our work provides an apparently novel condition for the validity of the QSSA in stochastic simulations of biochemical reaction networks with disparate timescales. PMID:25099817

Branched-chain amino acids and brain function.

PubMed

Fernstrom, John D

2005-06-01

Branched-chain amino acids (BCAAs) influence brain function by modifying large, neutral amino acid (LNAA) transport at the blood-brain barrier. Transport is shared by several LNAAs, notably the BCAAs and the aromatic amino acids (ArAAs), and is competitive. Consequently, when plasma BCAA concentrations rise, which can occur in response to food ingestion or BCAA administration, or with the onset of certain metabolic diseases (e.g., uncontrolled diabetes), brain BCAA concentrations rise, and ArAA concentrations decline. Such effects occur acutely and chronically. Such reductions in brain ArAA concentrations have functional consequences: biochemically, they reduce the synthesis and the release of neurotransmitters derived from ArAAs, notably serotonin (from tryptophan) and catecholamines (from tyrosine and phenylalanine). The functional effects of such neurochemical changes include altered hormonal function, blood pressure, and affective state. Although the BCAAs thus have biochemical and functional effects in the brain, few attempts have been made to characterize time-course or dose-response relations for such effects. And, no studies have attempted to identify levels of BCAA intake that might produce adverse effects on the brain. The only "model" of very high BCAA exposure is a very rare genetic disorder, maple syrup urine disease, a feature of which is substantial brain dysfunction but that probably cannot serve as a useful model for excessive BCAA intake by normal individuals. Given the known biochemical and functional effects of the BCAAs, it should be a straightforward exercise to design studies to assess dose-response relations for biochemical and functional effects and, in this context, to explore for adverse effect thresholds.

Multidimensional biochemical information processing of dynamical patterns

NASA Astrophysics Data System (ADS)

Hasegawa, Yoshihiko

2018-02-01

Cells receive signaling molecules by receptors and relay information via sensory networks so that they can respond properly depending on the type of signal. Recent studies have shown that cells can extract multidimensional information from dynamical concentration patterns of signaling molecules. We herein study how biochemical systems can process multidimensional information embedded in dynamical patterns. We model the decoding networks by linear response functions, and optimize the functions with the calculus of variations to maximize the mutual information between patterns and output. We find that, when the noise intensity is lower, decoders with different linear response functions, i.e., distinct decoders, can extract much information. However, when the noise intensity is higher, distinct decoders do not provide the maximum amount of information. This indicates that, when transmitting information by dynamical patterns, embedding information in multiple patterns is not optimal when the noise intensity is very large. Furthermore, we explore the biochemical implementations of these decoders using control theory and demonstrate that these decoders can be implemented biochemically through the modification of cascade-type networks, which are prevalent in actual signaling pathways.

Multidimensional biochemical information processing of dynamical patterns.

PubMed

Hasegawa, Yoshihiko

2018-02-01

Cells receive signaling molecules by receptors and relay information via sensory networks so that they can respond properly depending on the type of signal. Recent studies have shown that cells can extract multidimensional information from dynamical concentration patterns of signaling molecules. We herein study how biochemical systems can process multidimensional information embedded in dynamical patterns. We model the decoding networks by linear response functions, and optimize the functions with the calculus of variations to maximize the mutual information between patterns and output. We find that, when the noise intensity is lower, decoders with different linear response functions, i.e., distinct decoders, can extract much information. However, when the noise intensity is higher, distinct decoders do not provide the maximum amount of information. This indicates that, when transmitting information by dynamical patterns, embedding information in multiple patterns is not optimal when the noise intensity is very large. Furthermore, we explore the biochemical implementations of these decoders using control theory and demonstrate that these decoders can be implemented biochemically through the modification of cascade-type networks, which are prevalent in actual signaling pathways.

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...

CADLIVE toolbox for MATLAB: automatic dynamic modeling of biochemical networks with comprehensive system analysis.

PubMed

Inoue, Kentaro; Maeda, Kazuhiro; Miyabe, Takaaki; Matsuoka, Yu; Kurata, Hiroyuki

2014-09-01

Mathematical modeling has become a standard technique to understand the dynamics of complex biochemical systems. To promote the modeling, we had developed the CADLIVE dynamic simulator that automatically converted a biochemical map into its associated mathematical model, simulated its dynamic behaviors and analyzed its robustness. To enhance the feasibility by CADLIVE and extend its functions, we propose the CADLIVE toolbox available for MATLAB, which implements not only the existing functions of the CADLIVE dynamic simulator, but also the latest tools including global parameter search methods with robustness analysis. The seamless, bottom-up processes consisting of biochemical network construction, automatic construction of its dynamic model, simulation, optimization, and S-system analysis greatly facilitate dynamic modeling, contributing to the research of systems biology and synthetic biology. This application can be freely downloaded from http://www.cadlive.jp/CADLIVE_MATLAB/ together with an instruction.

Effects of subchronic extremely low-frequency electromagnetic field exposure on biochemical parameters in rats.

PubMed

Luo, Xue; Ma, Lingjuan; Gao, Peng; Zhang, Yanwen

2017-04-01

The objective of the present study was to systematically determine the effects of 50 Hertz (Hz) magnetic fields (MFs) on biochemical parameters in rats. Sixty-four adult (5 weeks old, 140-165 g) male Sprague-Dawley rats were randomly divided into four groups: sham, 20 µTesla (µT), 100 µT, and 500 µT 50 Hz MF ( n = 16 in each group). The rats in the MF groups were exposed for 2 h daily for up to 4 weeks. Under these experimental conditions, body weight, organ coefficients, biochemical parameters (blood lipids, myocardial enzymes, liver function, and renal function) were measured. We found that 50 Hz MFs had no significant effects on growth or on the majority of blood biochemical parameters, with the exception of creatinine and cholesterol. However, the changes in creatinine and cholesterol were relatively small and unlikely to be clinically relevant.

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

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.

The Glymphatic Hypothesis of Glaucoma: A Unifying Concept Incorporating Vascular, Biomechanical, and Biochemical Aspects of the Disease

PubMed Central

De Groot, Veva; Van Dam, Debby; Audenaert, Kurt; Killer, Hanspeter Esriel; De Deyn, Peter Paul

2017-01-01

The pathophysiology of primary open-angle glaucoma is still largely unknown, although a joint contribution of vascular, biomechanical, and biochemical factors is widely acknowledged. Since glaucoma is a leading cause of irreversible blindness worldwide, exploring its underlying pathophysiological mechanisms is extremely important and challenging. Evidence from recent studies appears supportive of the hypothesis that a “glymphatic system” exists in the eye and optic nerve, analogous to the described “glymphatic system” in the brain. As discussed in the present paper, elucidation of a glymphatic clearance pathway in the eye could provide a new unifying hypothesis of glaucoma that can incorporate many aspects of the vascular, biomechanical, and biochemical theories of the disease. It should be stressed, however, that the few research data currently available cannot be considered as proof of the existence of an “ocular glymphatic system” and that much more studies are needed to validate this possibility. Even though nothing conclusive can yet be said, the recent reports suggesting a paravascular transport system in the eye and optic nerve are encouraging and, if confirmed, may offer new perspectives for the development of novel diagnostic and therapeutic strategies for this devastating disorder. PMID:28948167

The Glymphatic Hypothesis of Glaucoma: A Unifying Concept Incorporating Vascular, Biomechanical, and Biochemical Aspects of the Disease.

PubMed

Wostyn, Peter; De Groot, Veva; Van Dam, Debby; Audenaert, Kurt; Killer, Hanspeter Esriel; De Deyn, Peter Paul

2017-01-01

The pathophysiology of primary open-angle glaucoma is still largely unknown, although a joint contribution of vascular, biomechanical, and biochemical factors is widely acknowledged. Since glaucoma is a leading cause of irreversible blindness worldwide, exploring its underlying pathophysiological mechanisms is extremely important and challenging. Evidence from recent studies appears supportive of the hypothesis that a "glymphatic system" exists in the eye and optic nerve, analogous to the described "glymphatic system" in the brain. As discussed in the present paper, elucidation of a glymphatic clearance pathway in the eye could provide a new unifying hypothesis of glaucoma that can incorporate many aspects of the vascular, biomechanical, and biochemical theories of the disease. It should be stressed, however, that the few research data currently available cannot be considered as proof of the existence of an "ocular glymphatic system" and that much more studies are needed to validate this possibility. Even though nothing conclusive can yet be said, the recent reports suggesting a paravascular transport system in the eye and optic nerve are encouraging and, if confirmed, may offer new perspectives for the development of novel diagnostic and therapeutic strategies for this devastating disorder.

Serum calprotectin levels correlate with biochemical and histological markers of disease activity in TNBS colitis

PubMed Central

Cury, Didia Bismara; Mizsputen, Sender Jankiel; Versolato, Clara; Miiji, Luciana Odashiro; Pereira, Edson; Delboni, Maria Aparecida; Schor, Nestor; Moss, Alan C.

2014-01-01

Background and aim Serum calprotectin is elevated in patients with inflammatory bowel disease (IBD). Whether it correlates other markers of disease activity is unknown. The aim of this study was to correlate serum calprotectin with biochemical and histological measures of intestinal inflammation. Materials and methods TNBS colitis was induced in wistar rats, and serial blood samples were collected at 0, 3, and 12 days. Animals were subsequently sacrificed for pathological evaluation at day 12. Serum calprotectin and cytokines were measured by ELISA. Pathologic changes were classified at the macroscopic and microscopic levels. Results TNBS colitis induced elevated serum calprotectin, TNF and IL-6 within 24 h. Levels of serum calprotectin remained elevated in parallel to persistence of loose stool and weight loss to day 12. Serum calprotectin levels correlated with serum levels of TNF-α and IL6 (p < 0.001), but not CRP. Animals with liquid stool had significantly higher levels of serum calprotectin than control animals. There was a correlation between macroscopic colitis scores, and levels of serum calprotectin. Conclusion Serum calprotectin levels correlate with biochemical and histological markers of inflammation in TNBS colitis. This biomarker may have potential for diagnostic use in patients with IBD. PMID:23685388

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

Iodoform gauze removes necrotic tissue from pressure ulcer wounds by fibrinolytic activity.

PubMed

Mizokami, Fumihiro; Murasawa, Yusuke; Furuta, Katsunori; Isogai, Zenzo

2012-01-01

Iodoform gauze is used in clinical practice for treatment of infected wounds. However, effectiveness and action mechanism of iodoform gauze for removal of necrotic tissue are unknown. We therefore employed case control and biochemical studies in order to clarify the pharmacological activity of iodoform gauze. A clinical study demonstrated that treatment with iodoform gauze removed necrotic tissue more effectively than treatment with conventional ointments. More than 60% of iodoform gauze-treated wounds were completely debrided within 2 weeks. Consistent with the clinical observation, biochemical analyses revealed clear differences in wound fluid proteins after treatment with iodoform gauze or conventional gauze. The amount of macroaggregates of type I collagen from wounds were remarkably decreased in iodoform gauze. Moreover, iodoform gauze and iodoform itself released non-aggregative type I collagen from necrotic debris in vitro. Taken together, we conclude that iodoform gauze efficiently removes necrotic tissue by its lytic activity for collagen fibers.

Maximum profile likelihood estimation of differential equation parameters through model based smoothing state estimates.

PubMed

Campbell, D A; Chkrebtii, O

2013-12-01

Statistical inference for biochemical models often faces a variety of characteristic challenges. In this paper we examine state and parameter estimation for the JAK-STAT intracellular signalling mechanism, which exemplifies the implementation intricacies common in many biochemical inference problems. We introduce an extension to the Generalized Smoothing approach for estimating delay differential equation models, addressing selection of complexity parameters, choice of the basis system, and appropriate optimization strategies. Motivated by the JAK-STAT system, we further extend the generalized smoothing approach to consider a nonlinear observation process with additional unknown parameters, and highlight how the approach handles unobserved states and unevenly spaced observations. The methodology developed is generally applicable to problems of estimation for differential equation models with delays, unobserved states, nonlinear observation processes, and partially observed histories. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

[Progresses of alpine treeline formation mechanism.

PubMed

Cong, Yu; He, Hong Shi; Gu, Xiao Nan; Xu, Wen Ru; Liu, Kai; Zong, Sheng Wei; Du, Hai Bo

2016-09-01

Alpine treelines represent one of the most distinct vegetation boundaries between canopy closed montane forest and treeless alpine vegetation. This transitional ecotone is highly sensitive to global and regional climate change and is considered as an ideal indicator of such changes. Treeline studies have evolved from morphological description to various hypotheses of treeline formation. Although individual hypothesis may provide reasonable explanation locally, a generalized hypothesis that is applicable on the global scale is still lacking. Temperature is considered the limited factor controlling the distribution of alpine treeline as low temperature restricts biochemical processes of tree growth. However, which particular biochemical processes are affected by low temperature remains unknown. This paper summarized the mechanisms of treeline formation with a focus on how low temperature affects photosynthesis characteristics, nutrient characteristics, non-structural carbohydrate (NSC) and antioxidant system. We also reviewed the key issues and future perspectives in treeline research.

Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

PubMed

Kerr, Ian D; Jones, Peter M; George, Anthony M

2010-02-01

One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

Biochemical functionalization of peptide nanotubes with phage displayed peptides

NASA Astrophysics Data System (ADS)

Swaminathan, Swathi; Cui, Yue

2016-09-01

The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

PubMed

Wu, Chenggang; Al Mamun, Abu Amar Mohamed; Luong, Truc Thanh; Hu, Bo; Gu, Jianhua; Lee, Ju Huck; D'Amore, Melissa; Das, Asis; Ton-That, Hung

2018-04-24

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the Δ ftsX and Δ envC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of Δ ftsX and Δ envC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated. IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum , due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the Δ ftsX and Δ envC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen. Copyright © 2018 Wu et al.

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella*

PubMed Central

Lin, Jianfeng; Tritschler, Douglas; Song, Kangkang; Barber, Cynthia F.; Cobb, Jennifer S.; Porter, Mary E.; Nicastro, Daniela

2011-01-01

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function. PMID:21700706

Effects of Biotin Deficiency on Biotinylated Proteins and Biotin-Related Genes in the Rat Brain.

PubMed

Yuasa, Masahiro; Aoyama, Yuki; Shimada, Ryoko; Sawamura, Hiromi; Ebara, Shuhei; Negoro, Munetaka; Fukui, Toru; Watanabe, Toshiaki

2016-01-01

Biotin is a water-soluble vitamin that functions as a cofactor for biotin-dependent carboxylases. The biochemical and physiological roles of biotin in brain regions have not yet been investigated sufficiently in vivo. Thus, in order to clarify the function of biotin in the brain, we herein examined biotin contents, biotinylated protein expression (e.g. holocarboxylases), and biotin-related gene expression in the brain of biotin-deficient rats. Three-week-old male Wistar rats were divided into a control group, biotin-deficient group, and pair-fed group. Rats were fed experimental diets from 3 wk old for 8 wk, and the cortex, hippocampus, striatum, hypothalamus, and cerebellum were then collected. In the biotin-deficient group, the maintenance of total biotin and holocarboxylases, increases in the bound form of biotin and biotinidase activity, and the expression of an unknown biotinylated protein were observed in the cortex. In other regions, total and free biotin contents decreased, holocarboxylase expression was maintained, and bound biotin and biotinidase activity remained unchanged. Biotin-related gene (pyruvate carboxylase, sodium-dependent multivitamin transporter, holocarboxylase synthetase, and biotinidase) expression in the cortex and hippocampus also remained unchanged among the dietary groups. These results suggest that biotin may be related to cortex functions by binding protein, and the effects of a biotin deficiency and the importance of biotin differ among the different brain regions.

Vfa1 binds to the N-terminal microtubule-interacting and trafficking (MIT) domain of Vps4 and stimulates its ATPase activity.

PubMed

Vild, Cody J; Xu, Zhaohui

2014-04-11

The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function.

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function*

PubMed Central

Brenig, Julian; de Boor, Susanne; Knyphausen, Philipp; Kuhlmann, Nora; Wroblowski, Sarah; Baldus, Linda; Scislowski, Lukas; Artz, Oliver; Trauschies, Philip; Baumann, Ulrich; Neundorf, Ines; Lammers, Michael

2015-01-01

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3. PMID:25911102

Do prokaryotes contain microtubules?

NASA Technical Reports Server (NTRS)

Bermudes, D.; Hinkle, G.; Margulis, L.

1994-01-01

In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin. They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement. Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established. Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules. Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes. We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown. At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules. In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells. Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins.

Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity*

PubMed Central

Vild, Cody J.; Xu, Zhaohui

2014-01-01

The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform[OPEN

PubMed Central

Nagarajan, Yagnesh; Rongala, Jay; Luang, Sukanya; Shadiac, Nadim; Sutton, Tim; Tyerman, Stephen D.; McPhee, Gordon; Voelcker, Nicolas H.; Lee, Jung-Goo

2016-01-01

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na+ ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na+-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na+ ion binding site. Our data enhance the understanding of the permeation functions of Bot1. PMID:26672067

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

PubMed

Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

2016-12-01

Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Isolated growth hormone deficiency (GHD) in childhood and adolescence: recent advances.

PubMed

Alatzoglou, Kyriaki S; Webb, Emma Alice; Le Tissier, Paul; Dattani, Mehul T

2014-06-01

The diagnosis of GH deficiency (GHD) in childhood is a multistep process involving clinical history, examination with detailed auxology, biochemical testing, and pituitary imaging, with an increasing contribution from genetics in patients with congenital GHD. Our increasing understanding of the factors involved in the development of somatotropes and the dynamic function of the somatotrope network may explain, at least in part, the development and progression of childhood GHD in different age groups. With respect to the genetic etiology of isolated GHD (IGHD), mutations in known genes such as those encoding GH (GH1), GHRH receptor (GHRHR), or transcription factors involved in pituitary development, are identified in a relatively small percentage of patients suggesting the involvement of other, yet unidentified, factors. Genome-wide association studies point toward an increasing number of genes involved in the control of growth, but their role in the etiology of IGHD remains unknown. Despite the many years of research in the area of GHD, there are still controversies on the etiology, diagnosis, and management of IGHD in children. Recent data suggest that childhood IGHD may have a wider impact on the health and neurodevelopment of children, but it is yet unknown to what extent treatment with recombinant human GH can reverse this effect. Finally, the safety of recombinant human GH is currently the subject of much debate and research, and it is clear that long-term controlled studies are needed to clarify the consequences of childhood IGHD and the long-term safety of its treatment.

X-Linked adrenoleukodystrophy is a frequent cause of idiopathic Addison`s disease in young adult male patients

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

Laureti, S.; Casucci, G.; Santeusanio, F.

1996-02-01

X-Linked adrenoleukodystrophy (ALD) is a genetic disease associated with demyelination of the central nervous system, adrenal insufficiency, and accumulation of very long chain fatty acids in tissue and body fluids. ALD is due to mutation of a gene located in Xq28 that encodes a peroxisomal transporter protein of unknown function. The most common phenotype of ALD is the cerebral form (45%) that develops in boys between 5-12 yr. Adrenomyeloneuropathy (AMN) involves the spinal cord and peripheral nerves in young adults (35%). Adrenal insufficiency (Addison`s disease) is frequently associated with AMN or cerebral ALD and may remain the only clinical expressionmore » of ALD (8% of cases). The prevalence of ALD among adults with Addison`s disease remains unknown. To evaluate this prevalence, we performed biochemical analysis of very long chain fatty acids in 14 male patients (age ranging from 12-45 yr at diagnosis) previously diagnosed as having primary idiopathic adrenocortical insufficiency. In 5 of 14 patients (35%), elevated plasma concentrations of very long chain fatty acids were detected. None of these patients had adrenocortical antibodies. By electrophysiological tests and magnetic resonance imaging it was determined that two patients had cerebral ALD, one had adrenomyeloneuropathy with cerebral involvement, and two had preclinical AMN. Our data support the hypothesis that ALD is a frequent cause of idiopathic Addison`s disease in children and adults. 30 refs., 5 tabs.« less

HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes.

PubMed

Morero, Natalia R; Botti, Horacio; Nitta, Kazuhiro R; Carrión, Federico; Obal, Gonzalo; Picardeau, Mathieu; Buschiazzo, Alejandro

2014-10-01

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role. © 2014 John Wiley & Sons Ltd.

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

Comprehensive functional characterization of the Glycoside Hydrolase Family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

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

Nelson, Cassandra E.; Attia, Mohamed A.; Rogowski, Artur

Here, lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1!3)/β(1!4) mixed-linkage glucan (MLG), and β(1!3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active Enzymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach we have delineated the physiological roles of the four C. japonicus Glycoside Hydrolase Family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activitymore » profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1!3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1!3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.« less

Comprehensive functional characterization of the Glycoside Hydrolase Family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

DOE PAGES

Nelson, Cassandra E.; Attia, Mohamed A.; Rogowski, Artur; ...

2017-10-20

Here, lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1!3)/β(1!4) mixed-linkage glucan (MLG), and β(1!3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active Enzymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach we have delineated the physiological roles of the four C. japonicus Glycoside Hydrolase Family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activitymore » profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1!3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1!3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.« less

Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification.

PubMed

Nelson, Cassandra E; Attia, Mohamed A; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G

2017-12-01

Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Interplay between environmentally modulated feedback loops - hypoxia and circadian rhythms - two sides of the same coin?

PubMed

Depping, Reinhard; Oster, Henrik

2017-11-01

Sensing of environmental parameters is critically important for cells of metazoan organisms. Members of the superfamily of bHLH-PAS transcription factors, involved in oxygen sensing and circadian rhythm generation, are important players in such molecular pathways. The interplay between both networks includes a so far unknown factor, connecting PER2 (circadian clocks) to hypoxia sensing (HIF-1 α) to result in a more adapted state of homeostasis at the right time. © 2017 Federation of European Biochemical Societies.

N-Acyl amino acids and their impact on biological processes.

PubMed

Hanuš, Lumír; Shohami, Esther; Bab, Itai; Mechoulam, Raphael

2014-01-01

Over the last two decades a large number of N-long-chain acyl amino acids have been identified in the mammalian body. The pharmacological activities of only a few of them have been investigated and some have been found to be of considerable interest. Thus arachidonoyl serine is vasodilatory and neuroprotective, arachidonoyl glycine is antinociceptive, and oleoyl serine rescues bone loss. However, the pathophysiological/biochemical roles of these amides are mostly unknown. © 2014 International Union of Biochemistry and Molecular Biology.

Relationship between changes in rat behavior and integral biochemical indexes determined by laser correlation spectroscopy after photothrombosis of the prefrontal cortex.

PubMed

Romanova, G A; Shakova, F M; Kovaleva, O I; Pivovarov, V V; Khlebnikova, N N; Karganov, M Yu

2004-02-01

Experiments on rats showed that Noopept improved retention and retrieval of conditioned passive avoidance response after phototrombosis of the prefrontal cortex (a procedure impairing retention of memory traces). The impairment of mnesic functions was accompanied by changes in integral biochemical indexes of the plasma determined by laser correlation spectroscopy. Treatment of behavioral disorders with Noopepet normalized biochemical indexes.

Spam1-associated transmission ratio distortion in mice: Elucidating the mechanism

PubMed Central

Martin-DeLeon, Patricia A; Zhang, Hong; Morales, Carlos R; Zhao, Yutong; Rulon, Michelle; Barnoski, Barry L; Chen, Hong; Galileo, Deni S

2005-01-01

Background While transmission ratio distortion, TRD, (a deviation from Mendelian ratio) is extensive in humans and well-documented in mice, the underlying mechanisms are unknown. Our earlier studies on carriers of spontaneous mutations of mouse Sperm Adhesion Molecule 1 (Spam1) suggested that TRD results from biochemically different sperm, due to a lack of transcript sharing through the intercellular cytoplasmic bridges of spermatids. These bridges usually allow transcript sharing among genetically different spermatids which develop into biochemically and functionally equivalent sperm. Objectives The goals of the study were to provide support for the lack of sharing (LOS) hypothesis, using transgene and null carriers of Spam1, and to determine the mechanism of Spam1-associated TRD. Methods Carriers of Spam1-Hyal5 BAC transgenes were mated with wild-type female mice and the progeny analyzed for TRD by PCR genotyping. Sperm from transgene and Spam1 null carriers were analyzed using flow cytometry and immunocytochemistry to detect quantities of Spam1 and/or Hyal5. Transgene-bearing sperm with Spam1 overexpression were detected by fluorescence in situ hybridization. In wild-type animals, EM studies of in situ transcript hybridization of testis sections and Northern analysis of biochemically fractionated testicular RNA were performed to localize Spam1 transcript. Finally, AU-rich motifs identified in the 3' UTR of Spam1 RNA were assayed by UV cross-linking to determine their ability to interact with testicular RNA binding proteins. Results The Tg8 line of transgene carriers had a significant (P < 0.001) TRD, due to reduced fertilizing ability of transgene-bearing sperm. These sperm retained large cytoplasmic droplets engorged with overexpressed Spam1 or Hyal5 protein. Caudal sperm from transgene carriers and caput sperm of null carriers showed a bimodal distribution of Spam1, indicating that the sperm in a male were biochemically different with respect to Spam1 quantities. Spam1 RNA was absent from the bridges, associated exclusively with the ER, and was shown to be anchored to the cytoskeleton. This compartmentalization of the transcript, mediated by cytoskeletal binding, occurs via protein interactions with 3' UTR AU-rich sequences that are likely involved in its stabilization. Conclusion We provide strong support for the LOS hypothesis, and have elucidated the mechanism of Spam1-associated TRD. PMID:16092963

Effects of carprofen on renal function and results of serum biochemical and hematologic analyses in anesthetized dogs that had low blood pressure during anesthesia.

PubMed

Boström, Ingrid M; Nyman, Görel C; Lord, Peter E; Häggström, Jens; Jones, Bernt E V; Bohlin, Henrik P

2002-05-01

To investigate effects of IV administered carprofen on indices of renal function and results of serum biochemical and hematologic analyses in dogs anesthetized with acepromazine-thiopentone-isoflurane that had low blood pressure during anesthesia. 6 healthy Beagles. A randomized crossover study was conducted, using the following treatments: saline (0.9% NaCl solution)-saline, saline-carprofen, and carprofen-saline. Saline (0.08 ml/kg) and carprofen (4 mg/kg) were administered IV. The first treatment was administered 30 minutes before induction of anesthesia and immediately before administration of acepromazine (0.1 mg/kg, IM). Anesthesia was induced with thiopentone (25 mg/ml, IV) and maintained with inspired isoflurane (2% in oxygen). The second treatment was administered 30 minutes after onset of inhalation anesthesia. Blood gases, circulation, and ventilation were monitored. Renal function was assessed by glomerular filtration rate (GFR), using scintigraphy, serum biochemical analyses, and urinalysis. Hematologic analysis was performed. Statistical analysis was conducted, using ANOVA or Friedman ANOVA. Values did not differ significantly among the 3 treatments. For all treatments, sedation and anesthesia caused changes in results of serum biochemical and hematologic analyses, a decrease in mean arterial blood pressure to 65 mm Hg, an increase of 115 pmol/L in angiotensin II concentration, and an increase of 100 seconds in time required to reach maximum activity counts during scintigraphy. Carprofen administered IV before or during anesthesia did not cause detectable significant adverse effects on renal function or results of serum biochemical and hematologic analyses in healthy Beagles with low blood pressure during anesthesia.

Ecological Functions of Landscapes

NASA Astrophysics Data System (ADS)

Kiryushin, V. I.

2018-01-01

Ecological functions of landscapes are considered a system of processes ensuring the development, preservation, and evolution of ecosystems and the biosphere as a whole. The concept of biogeocenosis can be considered a model that integrates biotic and environmental functions. The most general biogeocenotic functions specify the biodiversity, biotic links, self-organization, and evolution of ecosystems. Close interaction between biocenosis and the biotope (ecotope) is ensured by the continuous exchange of matter, energy, and information. Ecotope determines the biocenosis. The group of ecotopic functions includes atmospheric (gas exchange, heat exchange, hydroatmospheric, climate-forming), lithospheric (geodynamic, geophysical, and geochemical), hydrologic and hydrogeologic functions of landscape and ecotopic functions of soils. Bioecological functions emerge as a result of the biotope and ecotope interaction; these are the bioproductive, destructive, organoaccumulative, biochemical (gas, concentration, redox, biochemical, biopedological), pedogenetic, and energy functions

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.

Polyols in grape berry: transport and metabolic adjustments as a physiological strategy for water-deficit stress tolerance in grapevine.

PubMed

Conde, Artur; Regalado, Ana; Rodrigues, Diana; Costa, J Miguel; Blumwald, Eduardo; Chaves, M Manuela; Gerós, Hernâni

2015-02-01

Polyols are important metabolites that often function as carbon and energy sources and/or osmoprotective solutes in some plants. In grapevine, and in the grape berry in particular, the molecular aspects of polyol transport and metabolism and their physiological relevance are virtually unknown to date. Here, the biochemical function of a grapevine fruit mesocarp polyol transporter (VvPLT1) was characterized after its heterologous expression in yeast. This H(+)-dependent plasma membrane carrier transports mannitol (K m=5.4mM) and sorbitol (K m=9.5mM) over a broad range of polyols and monosaccharides. Water-deficit stress triggered an increase in the expression of VvPLT1 at the fully mature stage, allowing increased polyol uptake into pulp cells. Plant polyol dehydrogenases are oxireductases that reversibly oxidize polyols into monosaccharides. Mannitol catabolism in grape cells (K m=30.1mM mannitol) and mature berry mesocarps (K m=79mM) was, like sorbitol dehydrogenase activity, strongly inhibited (50-75%) by water-deficit stress. Simultaneously, fructose reduction into polyols via mannitol and sorbitol dehydrogenases was stimulated, contributing to their higher intracellular concentrations in water-deficit stress. Accordingly, the concentrations of mannitol, sorbitol, galactinol, myo-inositol, and dulcitol were significantly higher in berry mesocarps from water-deficit-stressed Tempranillo grapevines. Metabolomic profiling of the berry pulp by GC-TOF-MS also revealed many other changes in its composition induced by water deficit. The impact of polyols on grape berry composition and plant response to water deficit stress, via modifications in polyol transport and metabolism, was analysed by integrating metabolomics with transcriptional analysis and biochemical approaches. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Vitamin B-12 supplementation of rural Mexican women changes biochemical vitamin B-12 status indicators but does not affect hematology or a bone turnover marker.

PubMed

Shahab-Ferdows, Setareh; Anaya-Loyola, Miriam A; Vergara-Castañeda, Haydé; Rosado, Jorge L; Keyes, William R; Newman, John W; Miller, Joshua W; Allen, Lindsay H

2012-10-01

A high prevalence of low serum vitamin B-12 concentrations has been reported in studies and surveys in Latin America including Mexico, but the functional consequences are unknown. This randomized controlled trial assessed the response to a high-dose vitamin B-12 supplementation of women in rural Querétaro, Mexico. Participants aged 20-59 y were stratified at baseline to deficient, marginal, and adequate status groups (serum vitamin B-12, 75-148, 149-220, and >220 pmol/L, respectively), and each group was randomized to vitamin B-12 treatment (single dose of 1 mg i.m. then 500 μg/d orally for 3 mo, n = 70) or placebo (n = 62). Measures at baseline and 3 mo included: complete blood count, serum vitamin B-12, holotranscobalamin (holoTC), folate, ferritin, C-reactive protein (CRP), bone alkaline phosphatase, and methylmalonic acid (MMA) and plasma total homocysteine (tHcy). At baseline, 11% of the women were vitamin B-12 deficient and 22% had marginal status. HoloTC was low (<35 pmol/L) in 23% and correlated with serum vitamin B-12 (r = 0.7; P < 0.001). Elevated MMA (>271 nmol/L) and tHcy (>12 μmol/L) occurred in 21 and 31%, respectively, and correlated with serum vitamin B-12 (r = -0.28, P < 0.0007 and r = -0.20, P < 0.01, respectively). Supplementation increased serum vitamin B-12 and holoTC and lowered MMA and tHcy, normalizing all values except for elevated tHcy in 21% of the women. Supplementation did not affect hematology or bone-specific alkaline phosphatase. Vitamin B-12 supplementation normalized biochemical indicators of vitamin B-12 status in the treatment group but did not affect the functional outcomes measured.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231.

PubMed

Tenorio, María J; Ross, Breyan H; Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V; Ehrenfeld, Pamela; Mardones, Gonzalo A

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

PubMed Central

Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V.; Ehrenfeld, Pamela; Mardones, Gonzalo A.

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

PubMed Central

Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

2014-01-01

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID:25342745

Analysis of expression profiles of selected genes associated with the regenerative property and the receptivity to gene transfer during somatic embryogenesis in Triticum aestivum L.

PubMed

Delporte, Fabienne; Muhovski, Yordan; Pretova, Anna; Watillon, Bernard

2013-10-01

The physiological, biochemical and molecular mechanisms regulating the initiation of a regenerative pathway remain partially unknown. Efforts to identify the biological features that confer transformation ability, or the tendency of some cells to induce transgene silencing, would help to improve plant genetic engineering. The objective of our study was to monitor the evolution of plant cell competencies in relation to both in vitro tissue culture regeneration and the genetic transformation properties. We used a simple wheat regeneration procedure as an experimental model for studying the regenerative capacity of plant cells and their receptivity to direct gene transfer over the successive steps of the regenerative pathway. Target gene profiling studies and biochemical assays were conducted to follow some of the mechanisms triggered during the somatic-to-embryogenic transition (i.e. dedifferentiation, cell division activation, redifferentiation) and affecting the accessibility of plant cells to receive and stably express the exogenous DNA introduced by bombardment. Our results seem to indicate that the control of cell-cycle (S-phase) and host defense strategies can be crucial determinants of genetic transformation efficiency. The results from studies conducted at macro-, micro- and molecular scales are then integrated into a holistic approach that addresses the question of tissue culture and transgenesis competencies more broadly. Through this multilevel analysis we try to establish functional links between both regenerative capacity and transformation receptiveness, and thereby to provide a more global and integrated vision of both processes, at the core of defense/adaptive mechanisms and survival, between undifferentiated cell proliferation and organization.

Antidiabetic Activity and Chemical Composition of Sanbai Melon Seed Oil

PubMed Central

Li, Haili; Zhao, Hang; Zhang, Ya; Qiu, Pengcheng; Li, Jie

2018-01-01

Objectives Many fruits and herbs had been used in Traditional Chinese Medicines for treating diabetes mellitus (DM); however, scientific and accurate evidences regarding their efficacy and possible mechanisms were largely unknown. Sanbai melon seed oil (SMSO) was used in folk medicine in treating DM, but there is no literature about these effects. The present study was aimed at confirming the treatment effects of SMSO in type 1 DM. Methods Diabetes was induced by single intraperitoneal injection of streptozotocin (STZ) at a dose of 65 mg/kg body weight. After diabetes induction, mice were treated with SMSO at dose of 1 g/kg, 2 g/kg, and 4 g/kg. Drugs were given by gavage administration once a day continuously for 28 days. At the end of treatment, several biochemical parameters and molecular mechanisms were determined by biochemical assays, ELISA, and Western blotting. The chemical compositions of SMSO were also tested. Results SMSO treatment significantly improved the symptoms of weight loss, polydipsia, reduced FBG level, increased plasma insulin levels, reduced plasma lipids levels, and protected islet injury. The results also showed that SMSO mitigated oxidative stress and alleviated the liver and renal injury in diabetes mice. SMSO also protected islet cells from apoptotic damage by suppressing ER mediated and mitochondrial dependent apoptotic pathways. Further constituent analysis results showed that SMSO had rich natural resources which had beneficial effects on DM. Conclusions This study showed that SMSO had excellent antidiabetes effect and provided scientific basis for the use of SMSO as the functional ingredients production and dietary supplements production in the food and pharmaceutical industries. PMID:29853958

Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

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

Jin, R.; Sikorra, S.; Stegmann, C.M.

2009-06-01

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognitionmore » and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.« less

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

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

Stanek, Kimberly A.; Patterson-West, Jennifer; Randolph, Peter S.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homologmore » has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U 6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.« less

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

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.

Neuron-specific caveolin-1 overexpression improves motor function and preserves memory in mice subjected to brain trauma.

PubMed

Egawa, Junji; Schilling, Jan M; Cui, Weihua; Posadas, Edmund; Sawada, Atsushi; Alas, Basheer; Zemljic-Harpf, Alice E; Fannon-Pavlich, McKenzie J; Mandyam, Chitra D; Roth, David M; Patel, Hemal H; Patel, Piyush M; Head, Brian P

2017-08-01

Studies in vitro and in vivo demonstrate that membrane/lipid rafts and caveolin (Cav) organize progrowth receptors, and, when overexpressed specifically in neurons, Cav-1 augments neuronal signaling and growth and improves cognitive function in adult and aged mice; however, whether neuronal Cav-1 overexpression can preserve motor and cognitive function in the brain trauma setting is unknown. Here, we generated a neuron-targeted Cav-1-overexpressing transgenic (Tg) mouse [synapsin-driven Cav-1 (SynCav1 Tg)] and subjected it to a controlled cortical impact model of brain trauma and measured biochemical, anatomic, and behavioral changes. SynCav1 Tg mice exhibited increased hippocampal expression of Cav-1 and membrane/lipid raft localization of postsynaptic density protein 95, NMDA receptor, and tropomyosin receptor kinase B. When subjected to a controlled cortical impact, SynCav1 Tg mice demonstrated preserved hippocampus-dependent fear learning and memory, improved motor function recovery, and decreased brain lesion volume compared with wild-type controls. Neuron-targeted overexpression of Cav-1 in the adult brain prevents hippocampus-dependent learning and memory deficits, restores motor function after brain trauma, and decreases brain lesion size induced by trauma. Our findings demonstrate that neuron-targeted Cav-1 can be used as a novel therapeutic strategy to restore brain function and prevent trauma-associated maladaptive plasticity.-Egawa, J., Schilling, J. M., Cui, W., Posadas, E., Sawada, A., Alas, B., Zemljic-Harpf, A. E., Fannon-Pavlich, M. J., Mandyam, C. D., Roth, D. M., Patel, H. H., Patel, P. M., Head, B. P. Neuron-specific caveolin-1 overexpression improves motor function and preserves memory in mice subjected to brain trauma. © FASEB.

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.

Approximation-based adaptive tracking control of pure-feedback nonlinear systems with multiple unknown time-varying delays.

PubMed

Wang, Min; Ge, Shuzhi Sam; Hong, Keum-Shik

2010-11-01

This paper presents adaptive neural tracking control for a class of non-affine pure-feedback systems with multiple unknown state time-varying delays. To overcome the design difficulty from non-affine structure of pure-feedback system, mean value theorem is exploited to deduce affine appearance of state variables x(i) as virtual controls α(i), and of the actual control u. The separation technique is introduced to decompose unknown functions of all time-varying delayed states into a series of continuous functions of each delayed state. The novel Lyapunov-Krasovskii functionals are employed to compensate for the unknown functions of current delayed state, which is effectively free from any restriction on unknown time-delay functions and overcomes the circular construction of controller caused by the neural approximation of a function of u and [Formula: see text] . Novel continuous functions are introduced to overcome the design difficulty deduced from the use of one adaptive parameter. To achieve uniformly ultimate boundedness of all the signals in the closed-loop system and tracking performance, control gains are effectively modified as a dynamic form with a class of even function, which makes stability analysis be carried out at the present of multiple time-varying delays. Simulation studies are provided to demonstrate the effectiveness of the proposed scheme.

Existence conditions for unknown input functional observers

NASA Astrophysics Data System (ADS)

Fernando, T.; MacDougall, S.; Sreeram, V.; Trinh, H.

2013-01-01

This article presents necessary and sufficient conditions for the existence and design of an unknown input Functional observer. The existence of the observer can be verified by computing a nullspace of a known matrix and testing some matrix rank conditions. The existence of the observer does not require the satisfaction of the observer matching condition (i.e. Equation (16) in Hou and Muller 1992, 'Design of Observers for Linear Systems with Unknown Inputs', IEEE Transactions on Automatic Control, 37, 871-875), is not limited to estimating scalar functionals and allows for arbitrary pole placement. The proposed observer always exists when a state observer exists for the unknown input system, and furthermore, the proposed observer can exist even in some instances when an unknown input state observer does not exist.

Identification of cadmium-induced Agaricus blazei genes through suppression subtractive hybridization.

PubMed

Wang, Liling; Li, Haibo; Wei, Hailong; Wu, Xueqian; Ke, Leqin

2014-01-01

Cadmium (Cd) is one of the most serious environmental pollutants. Filamentous fungi are very promising organisms for controlling and reducing the amount of heavy metals released by human and industrial activities. However, the molecular mechanisms involved in Cd accumulation and tolerance of filamentous fungi are not fully understood. Agaricus blazei Murrill, an edible mushroom with medicinal properties, demonstrates high tolerance for heavy metals, especially Cd. To investigate the molecular mechanisms underlying the response of A. blazei after Cd exposure, we constructed a forward subtractive library that represents cadmium-induced genes in A. blazei under 4 ppm Cd stress for 14 days using suppression subtractive hybridization combined with mirror orientation selection. Differential screening allowed us to identify 39 upregulated genes, 26 of which are involved in metabolism, protein fate, cellular transport, transport facilitation and transport routes, cell rescue, defense and virulence, transcription, and the action of proteins with a binding function, and 13 are encoding hypothetical proteins with unknown functions. Induction of six A. blazei genes after Cd exposure was further confirmed by RT-qPCR. The cDNAs isolated in this study contribute to our understanding of genes involved in the biochemical pathways that participate in the response of filamentous fungi to Cd exposure. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

A Mental Retardation-linked Nonsense Mutation in Cereblon Is Rescued by Proteasome Inhibition*

PubMed Central

Xu, Guoqiang; Jiang, Xiaogang; Jaffrey, Samie R.

2013-01-01

A nonsense mutation in cereblon (CRBN) causes autosomal recessive nonsyndromic mental retardation. Cereblon is a substrate receptor for the Cullin-RING E3 ligase complex and couples the ubiquitin ligase to specific ubiquitination targets. The CRBN nonsense mutation (R419X) results in a protein lacking 24 amino acids at its C terminus. Although this mutation has been linked to mild mental retardation, the mechanism by which the mutation affects CRBN function is unknown. Here, we used biochemical and mass spectrometric approaches to explore the function of this mutant. We show that the protein retains its ability to assemble into a Cullin-RING E3 ligase complex and catalyzes the ubiquitination of CRBN-target proteins. However, we find that this mutant exhibits markedly increased levels of autoubiquitination and is more readily degraded by the proteasome than the wild type protein. We also show that the level of the mutant protein can be restored by a treatment of cells with a clinically utilized proteasome inhibitor, suggesting that this agent may be useful for the treatment of mental retardation associated with the CRBN R419X mutation. These data demonstrate that enhanced autoubiquitination and degradation account for the defect in CRBN activity that leads to mental retardation. PMID:23983124

Identification of genes differentially expressed in ectomycorrhizal roots during the Pinus pinaster-Laccaria bicolor interaction.

PubMed

Flores-Monterroso, Aranzazu; Canales, Javier; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

2013-06-01

Ectomycorrhizal associations are of major ecological importance in temperate and boreal forests. The development of a functional ectomycorrhiza requires many genetic and biochemical changes. In this study, suppressive subtraction hybridization was used to identify differentially expressed genes in the roots of maritime pine (Pinus pinaster Aiton) inoculated with Laccaria bicolor, a mycorrhizal fungus. A total number of 200 unigenes were identified as being differentially regulated in maritime pine roots during the development of mycorrhiza. These unigenes were classified into 10 categories according to the function of their homologues in the GenBank database. Approximately, 40 % of the differentially expressed transcripts were genes that coded for unknown proteins in the databases or that had no homology to known genes. A group of these differentially expressed genes was selected to validate the results using quantitative real-time PCR. The transcript levels of the representative genes were compared between the non-inoculated and inoculated plants at 1, 5, 15 and 30 days after inoculation. The observed expression patterns indicate (1) changes in the composition of the wall cell, (2) tight regulation of defence genes during the development of mycorrhiza and (3) changes in carbon and nitrogen metabolism. Ammonium excess or deficiency dramatically affected the stability of ectomycorrhiza and altered gene expression in maritime pine roots.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

Leaf proteomic analysis in cassava (Manihot esculenta, Crantz) during plant development, from planting of stem cutting to storage root formation.

PubMed

Mitprasat, Mashamon; Roytrakul, Sittiruk; Jiemsup, Surasak; Boonseng, Opas; Yokthongwattana, Kittisak

2011-06-01

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student's t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC-MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.

Blocking Effects of Human Tau on Squid Giant Synapse Transmission and Its Prevention by T-817 MA

PubMed Central

Moreno, Herman; Choi, Soonwook; Yu, Eunah; Brusco, Janaina; Avila, Jesus; Moreira, Jorge E.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

2011-01-01

Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity. PMID:21629767

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

PubMed

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

*CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

PubMed Central

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data shows that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture show that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data shows that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation, and suggests that Emp may be involved in multiple cellular functions. PMID:17071116

Malaria Parasite CLAG3, a Protein Linked to Nutrient Channels, Participates in High Molecular Weight Membrane-Associated Complexes in the Infected Erythrocyte

PubMed Central

Zainabadi, Kayvan

2016-01-01

Malaria infected erythrocytes show increased permeability to a number of solutes important for parasite growth as mediated by the Plasmodial Surface Anion Channel (PSAC). The P. falciparum clag3 genes have recently been identified as key determinants of PSAC, though exactly how they contribute to channel function and whether additional host/parasite proteins are required remain unknown. To begin to answer these questions, I have taken a biochemical approach. Here I have used an epitope-tagged CLAG3 parasite to perform co-immunoprecipitation experiments using membrane fractions of infected erythrocytes. Native PAGE and mass spectrometry studies reveal that CLAG3 participate in at least three different high molecular weight complexes: a ~720kDa complex consisting of CLAG3, RHOPH2 and RHOPH3; a ~620kDa complex consisting of CLAG3 and RHOPH2; and a ~480kDa complex composed solely of CLAG3. Importantly, these complexes can be found throughout the parasite lifecycle but are absent in untransfected controls. Extracellular biotin labeling and protease susceptibility studies localize the 480kDa complex to the erythrocyte membrane. This complex, likely composed of a homo-oligomer of 160kDa CLAG3, may represent a functional subunit, possibly the pore, of PSAC. PMID:27299521

Muscular Dystrophy with Ribitol-Phosphate Deficiency: A Novel Post-Translational Mechanism in Dystroglycanopathy

PubMed Central

Kanagawa, Motoi; Toda, Tatsushi

2017-01-01

Muscular dystrophy is a group of genetic disorders characterized by progressive muscle weakness. In the early 2000s, a new classification of muscular dystrophy, dystroglycanopathy, was established. Dystroglycanopathy often associates with abnormalities in the central nervous system. Currently, at least eighteen genes have been identified that are responsible for dystroglycanopathy, and despite its genetic heterogeneity, its common biochemical feature is abnormal glycosylation of alpha-dystroglycan. Abnormal glycosylation of alpha-dystroglycan reduces its binding activities to ligand proteins, including laminins. In just the last few years, remarkable progress has been made in determining the sugar chain structures and gene functions associated with dystroglycanopathy. The normal sugar chain contains tandem structures of ribitol-phosphate, a pentose alcohol that was previously unknown in humans. The dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and isoprenoid synthase domain-containing protein (ISPD) encode essential enzymes for the synthesis of this structure: fukutin and FKRP transfer ribitol-phosphate onto sugar chains of alpha-dystroglycan, and ISPD synthesizes CDP-ribitol, a donor substrate for fukutin and FKRP. These findings resolved long-standing questions and established a disease subgroup that is ribitol-phosphate deficient, which describes a large population of dystroglycanopathy patients. Here, we review the history of dystroglycanopathy, the properties of the sugar chain structure of alpha-dystroglycan, dystroglycanopathy gene functions, and therapeutic strategies. PMID:29081423

Improved biochemical preservation of heart slices during cold storage.

PubMed

Bull, D A; Reid, B B; Connors, R C; Albanil, A; Stringham, J C; Karwande, S V

2000-01-01

Development of myocardial preservation solutions requires the use of whole organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that myocardial slices could be used to assess preservation of biochemical function during cold storage. Whole rat hearts were precision cut into slices with a thickness of 200 microm and preserved at 4 degrees C in one of the following solutions: Columbia University (CU), University of Wisconsin (UW), D5 0.2% normal saline with 20 meq/l KCL (QNS), normal saline (NS), or a novel cardiac preservation solution (NPS) developed using this model. Myocardial biochemical function was assessed by ATP content (etamoles ATP/mg wet weight) and capacity for protein synthesis (counts per minute (cpm)/mg protein) immediately following slicing (0 hours), and at 6, 12, 18, and 24 hours of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as the mean +/- standard deviation. ATP content was higher in the heart slices stored in the NPS compared to all other solutions at 6, 12, 18 and 24 hours of cold storage (p < 0.05). Capacity for protein synthesis was higher in the heart slices stored in the NPS compared to all other solutions at 6, 12, and 18 hours of cold storage (p < 0.05). CONCLUSIONS This myocardial slice model allows the rapid and efficient screening of cardiac preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel preservation solution which improves the biochemical function of myocardial slices during cold storage.

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

A Unified Approach to Adaptive Neural Control for Nonlinear Discrete-Time Systems With Nonlinear Dead-Zone Input.

PubMed

Liu, Yan-Jun; Gao, Ying; Tong, Shaocheng; Chen, C L Philip

2016-01-01

In this paper, an effective adaptive control approach is constructed to stabilize a class of nonlinear discrete-time systems, which contain unknown functions, unknown dead-zone input, and unknown control direction. Different from linear dead zone, the dead zone, in this paper, is a kind of nonlinear dead zone. To overcome the noncausal problem, which leads to the control scheme infeasible, the systems can be transformed into a m -step-ahead predictor. Due to nonlinear dead-zone appearance, the transformed predictor still contains the nonaffine function. In addition, it is assumed that the gain function of dead-zone input and the control direction are unknown. These conditions bring about the difficulties and the complicacy in the controller design. Thus, the implicit function theorem is applied to deal with nonaffine dead-zone appearance, the problem caused by the unknown control direction can be resolved through applying the discrete Nussbaum gain, and the neural networks are used to approximate the unknown function. Based on the Lyapunov theory, all the signals of the resulting closed-loop system are proved to be semiglobal uniformly ultimately bounded. Moreover, the tracking error is proved to be regulated to a small neighborhood around zero. The feasibility of the proposed approach is demonstrated by a simulation example.

Proteins of unknown function in the Protein Data Bank (PDB): an inventory of true uncharacterized proteins and computational tools for their analysis.

PubMed

Nadzirin, Nurul; Firdaus-Raih, Mohd

2012-10-08

Proteins of uncharacterized functions form a large part of many of the currently available biological databases and this situation exists even in the Protein Data Bank (PDB). Our analysis of recent PDB data revealed that only 42.53% of PDB entries (1084 coordinate files) that were categorized under "unknown function" are true examples of proteins of unknown function at this point in time. The remainder 1465 entries also annotated as such appear to be able to have their annotations re-assessed, based on the availability of direct functional characterization experiments for the protein itself, or for homologous sequences or structures thus enabling computational function inference.

Writing and compiling code into biochemistry.

PubMed

Shea, Adam; Fett, Brian; Riedel, Marc D; Parhi, Keshab

2010-01-01

This paper presents a methodology for translating iterative arithmetic computation, specified as high-level programming constructs, into biochemical reactions. From an input/output specification, we generate biochemical reactions that produce output quantities of proteins as a function of input quantities performing operations such as addition, subtraction, and scalar multiplication. Iterative constructs such as "while" loops and "for" loops are implemented by transferring quantities between protein types, based on a clocking mechanism. Synthesis first is performed at a conceptual level, in terms of abstract biochemical reactions - a task analogous to high-level program compilation. Then the results are mapped onto specific biochemical reactions selected from libraries - a task analogous to machine language compilation. We demonstrate our approach through the compilation of a variety of standard iterative functions: multiplication, exponentiation, discrete logarithms, raising to a power, and linear transforms on time series. The designs are validated through transient stochastic simulation of the chemical kinetics. We are exploring DNA-based computation via strand displacement as a possible experimental chassis.

Design of a biochemical circuit motif for learning linear functions

PubMed Central

Lakin, Matthew R.; Minnich, Amanda; Lane, Terran; Stefanovic, Darko

2014-01-01

Learning and adaptive behaviour are fundamental biological processes. A key goal in the field of bioengineering is to develop biochemical circuit architectures with the ability to adapt to dynamic chemical environments. Here, we present a novel design for a biomolecular circuit capable of supervised learning of linear functions, using a model based on chemical reactions catalysed by DNAzymes. To achieve this, we propose a novel mechanism of maintaining and modifying internal state in biochemical systems, thereby advancing the state of the art in biomolecular circuit architecture. We use simulations to demonstrate that the circuit is capable of learning behaviour and assess its asymptotic learning performance, scalability and robustness to noise. Such circuits show great potential for building autonomous in vivo nanomedical devices. While such a biochemical system can tell us a great deal about the fundamentals of learning in living systems and may have broad applications in biomedicine (e.g. autonomous and adaptive drugs), it also offers some intriguing challenges and surprising behaviours from a machine learning perspective. PMID:25401175

Design of a biochemical circuit motif for learning linear functions.

PubMed

Lakin, Matthew R; Minnich, Amanda; Lane, Terran; Stefanovic, Darko

2014-12-06

Learning and adaptive behaviour are fundamental biological processes. A key goal in the field of bioengineering is to develop biochemical circuit architectures with the ability to adapt to dynamic chemical environments. Here, we present a novel design for a biomolecular circuit capable of supervised learning of linear functions, using a model based on chemical reactions catalysed by DNAzymes. To achieve this, we propose a novel mechanism of maintaining and modifying internal state in biochemical systems, thereby advancing the state of the art in biomolecular circuit architecture. We use simulations to demonstrate that the circuit is capable of learning behaviour and assess its asymptotic learning performance, scalability and robustness to noise. Such circuits show great potential for building autonomous in vivo nanomedical devices. While such a biochemical system can tell us a great deal about the fundamentals of learning in living systems and may have broad applications in biomedicine (e.g. autonomous and adaptive drugs), it also offers some intriguing challenges and surprising behaviours from a machine learning perspective.

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.

Functional Proteomic Analysis of Signaling Networks and Response to Targeted Therapy

DTIC Science & Technology

2009-03-01

of biological networks. Nature Biotechnology, 23(9):961–966, 2005. [18] A. Ma’ayan, S. L Jenkins, S. Neves, A. Hasseldine, E. Grace, B . Dubin-Thaler...functions of biochemical networks. Trends Biochemical Sci 31: 284–291. 56. Blinov ML, Faeder JR, Goldstein B , Hlavacek WS (2006) A network model of early...mean intensity value, red - increased intensity of signal and green - decreased intensity of signal. Lap- Lapatinib, Das- Dasatinib, C-control, A& B

Induction of ovarian function by using short-term human menopausal gonadotrophin in patients with ovarian failure following cytotoxic chemotherapy for haematological malignancy.

PubMed

Chatterjee, R; Mills, W; Katz, M; McGarrigle, H H; Goldstone, A H

1993-07-01

Currently no treatment has proved successful in inducing ovarian steroidogenic and/or gametogenic recovery in patients with haematological malignancies treated by cytotoxic chemotherapy once biochemical failure becomes manifest i.e., when FSH levels exceed 40 IU/L. This paper reports two such cases with classical biochemical ovarian failure in which ovarian function was induced by brief stimulation with Human Menopausal Gonadotrophin (HMG).

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.

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

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl-tRNA synthetase activity.

PubMed

Comisso, Martine; Hadchouel, Alice; de Blic, Jacques; Mirande, Marc

2018-05-18

Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNA Met -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP. © 2018 Federation of European Biochemical Societies.

Mitotic Dysfunction Associated with Aging Hallmarks.

PubMed

Macedo, Joana Catarina; Vaz, Sara; Logarinho, Elsa

2017-01-01

Aging is a biological process characterized by the progressive deterioration of physiological functions known to be the main risk factor for chronic diseases and declining health. There has been an emerging connection between aging and aneuploidy, an aberrant number of chromosomes, even though the molecular mechanisms behind age-associated aneuploidy remain largely unknown. In recent years, several genetic pathways and biochemical processes controlling the rate of aging have been identified and proposed as aging hallmarks. Primary hallmarks that cause the accumulation of cellular damage include genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis (López-Otín et al., Cell 153:1194-1217, 2013). Here we review the provocative link between these aging hallmarks and the loss of chromosome segregation fidelity during cell division, which could support the correlation between aging and aneuploidy seen over the past decades. Secondly, we review the systemic impacts of aneuploidy in cell physiology and emphasize how these include some of the primary hallmarks of aging. Based on the evidence, we propose a mutual causality between aging and aneuploidy, and suggest modulation of mitotic fidelity as a potential means to ameliorate healthy lifespan.

The binding, transport and fate of aluminium in biological cells.

PubMed

Exley, Christopher; Mold, Matthew J

2015-04-01

Aluminium is the most abundant metal in the Earth's crust and yet, paradoxically, it has no known biological function. Aluminium is biochemically reactive, it is simply that it is not required for any essential process in extant biota. There is evidence neither of element-specific nor evolutionarily conserved aluminium biochemistry. This means that there are no ligands or chaperones which are specific to its transport, there are no transporters or channels to selectively facilitate its passage across membranes, there are no intracellular storage proteins to aid its cellular homeostasis and there are no pathways which evolved to enable the metabolism and excretion of aluminium. Of course, aluminium is found in every compartment of every cell of every organism, from virus through to Man. Herein we have investigated each of the 'silent' pathways and metabolic events which together constitute a form of aluminium homeostasis in biota, identifying and evaluating as far as is possible what is known and, equally importantly, what is unknown about its uptake, transport, storage and excretion. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

Malet, Hélène; Dalle, Karen; Brémond, Nicolas

The SARS-CoV macro domain was expressed, purified and crystallized. Selenomethionine-labelled crystals diffracted to 1.8 Å resolution. Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1′′-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macromore » domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182–355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 Å, β = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 Å.« less

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases

PubMed Central

Bradshaw, Niels; Levdikov, Vladimir M; Zimanyi, Christina M; Gaudet, Rachelle; Wilkinson, Anthony J; Losick, Richard

2017-01-01

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. DOI: http://dx.doi.org/10.7554/eLife.26111.001 PMID:28527238

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

PubMed

Schiklenk, Christoph; Petrova, Boryana; Kschonsak, Marc; Hassler, Markus; Klein, Carlo; Gibson, Toby J; Haering, Christian H

2018-05-07

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C 2 H 2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation. © 2018 Schiklenk et al.

Structural basis for Mob1-dependent activation of the core Mst–Lats kinase cascade in Hippo signaling

DOE PAGES

Ni, Lisheng; Zheng, Yonggang; Hara, Mayuko; ...

2015-06-24

The Mst–Lats kinase cascade is central to the Hippo tumor-suppressive pathway that controls organ size and tissue homeostasis. The adaptor protein Mob1 promotes Lats activation by Mst, but the mechanism remains unknown. Here, we show that human Mob1 binds to autophosphorylated docking motifs in active Mst2. This binding enables Mob1 phosphorylation by Mst2. Phosphorylated Mob1 undergoes conformational activation and binds to Lats1. We determine the crystal structures of phospho-Mst2–Mob1 and phospho-Mob1–Lats1 complexes, revealing the structural basis of both phosphorylation-dependent binding events. Further biochemical and functional analyses demonstrate that Mob1 mediates Lats1 activation through dynamic scaffolding and allosteric mechanisms. Thus, Mob1more » acts as a phosphorylation-regulated coupler of kinase activation by virtue of its ability to engage multiple ligands. We propose that stepwise, phosphorylation-triggered docking interactions of nonkinase elements enhance the specificity and robustness of kinase signaling cascades.« less

Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

PubMed

Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

2015-12-01

Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

PubMed

Zhang, Nan; Han, Zhentai; Sun, Guiling; Hoffman, Angela; Wilson, Iain W; Yang, Yanfang; Gao, Qian; Wu, Jianqiang; Xie, Dan; Dai, Jungui; Qiu, Deyou

2014-01-17

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp. Copyright © 2013 Elsevier Inc. All rights reserved.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

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

Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation.

PubMed

Ho, Chai-Ling; Teoh, Seddon; Teo, Swee-Sen; Rahim, Raha Abdul; Phang, Siew-Moi

2009-01-01

Light regulates photosynthesis, growth and reproduction, yield and properties of phycocolloids, and starch contents in seaweeds. Despite its importance as an environmental cue that regulates many developmental, physiological, and biochemical processes, the network of genes involved during light deprivation are obscure. In this study, we profiled the transcriptome of Gracilaria changii at two different irradiance levels using a cDNA microarray containing more than 3,000 cDNA probes. Microarray analysis revealed that 93 and 105 genes were up- and down-regulated more than 3-fold under light deprivation, respectively. However, only 50% of the transcripts have significant matches to the nonredundant peptide sequences in the database. The transcripts that accumulated under light deprivation include vanadium chloroperoxidase, thioredoxin, ferredoxin component, and reduced nicotinamide adenine dinucleotide dehydrogenase. Among the genes that were down-regulated under light deprivation were genes encoding light harvesting protein, light harvesting complex I, phycobilisome 7.8 kDa linker polypeptide, low molecular weight early light-inducible protein, and vanadium bromoperoxidase. Our findings also provided important clues to the functions of many unknown sequences that could not be annotated using sequence comparison.

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

PubMed Central

Xie, Xingqiao; Li, Faxiang; Wang, Yuanyuan; Wang, Yingli; Lin, Zhijie; Cheng, Xiaofang; Liu, Jianping; Chen, Changbin; Pan, Lifeng

2015-01-01

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations. PMID:26506893

Ab Initio Structural Modeling of and Experimental Validation for Chlamydia trachomatis Protein CT296 Reveal Structural Similarity to Fe(II) 2-Oxoglutarate-Dependent Enzymes▿

PubMed Central

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott; Battaile, Kevin P.; Zhang, Yang; Hefty, P. Scott

2011-01-01

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF) CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-Å Cα root mean square deviation [RMSD]) the high-resolution (1.8-Å) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur. PMID:21965559

Ab initio structural modeling of and experimental validation for Chlamydia trachomatis protein CT296 reveal structural similarity to Fe(II) 2-oxoglutarate-dependent enzymes

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

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott

2012-02-13

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF)more » CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-{angstrom} C{alpha} root mean square deviation [RMSD]) the high-resolution (1.8-{angstrom}) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur.« less

Evaluation method for the potential functionome harbored in the genome and metagenome.

PubMed

Takami, Hideto; Taniguchi, Takeaki; Moriya, Yuki; Kuwahara, Tomomi; Kanehisa, Minoru; Goto, Susumu

2012-12-12

One of the main goals of genomic analysis is to elucidate the comprehensive functions (functionome) in individual organisms or a whole community in various environments. However, a standard evaluation method for discerning the functional potentials harbored within the genome or metagenome has not yet been established. We have developed a new evaluation method for the potential functionome, based on the completion ratio of Kyoto Encyclopedia of Genes and Genomes (KEGG) functional modules. Distribution of the completion ratio of the KEGG functional modules in 768 prokaryotic species varied greatly with the kind of module, and all modules primarily fell into 4 patterns (universal, restricted, diversified and non-prokaryotic modules), indicating the universal and unique nature of each module, and also the versatility of the KEGG Orthology (KO) identifiers mapped to each one. The module completion ratio in 8 phenotypically different bacilli revealed that some modules were shared only in phenotypically similar species. Metagenomes of human gut microbiomes from 13 healthy individuals previously determined by the Sanger method were analyzed based on the module completion ratio. Results led to new discoveries in the nutritional preferences of gut microbes, believed to be one of the mutualistic representations of gut microbiomes to avoid nutritional competition with the host. The method developed in this study could characterize the functionome harbored in genomes and metagenomes. As this method also provided taxonomical information from KEGG modules as well as the gene hosts constructing the modules, interpretation of completion profiles was simplified and we could identify the complementarity between biochemical functions in human hosts and the nutritional preferences in human gut microbiomes. Thus, our method has the potential to be a powerful tool for comparative functional analysis in genomics and metagenomics, able to target unknown environments containing various uncultivable microbes within unidentified phyla.

Biochemical Activities of 320 ToxCast Chemicals Evaluated Across 239 Functional Targets

EPA Science Inventory

EPA’s ToxCast research program is profiling chemical bioactivity in order to generate predictive signatures of toxicity. The present study evaluated 320 chemicals across 239 biochemical assays. ToxCast phase I chemicals include 309 unique structures, most of which are pesticide ...

Cobalamin supplementation improves motor development and regurgitations in infants: results from a randomized intervention study.

PubMed

Torsvik, Ingrid; Ueland, Per Magne; Markestad, Trond; Bjørke-Monsen, Anne-Lise

2013-11-01

During infancy, minor developmental delays and gastrointestinal complaints are common, as is a biochemical profile indicative of impaired cobalamin status. We investigated whether cobalamin supplementation can improve development or symptoms in infants with biochemical signs of impaired cobalamin function and developmental delay or feeding difficulties. Infants <8 mo of age (n = 105) who were referred for feeding difficulties, subtle neurologic symptoms, or delayed psychomotor development were assessed for cobalamin status [by the measurement of serum cobalamin, plasma total homocysteine (tHcy), and plasma methylmalonic acid (MMA)]. Infants with biochemical signs of impaired cobalamin function, defined as a plasma tHcy concentration ≥6.5 μmol/L (n = 79), were enrolled in a double-blind, randomized controlled trial to receive 400 μg hydroxycobalamin intramuscularly (n = 42) or a sham injection (n = 37). Motor function [Alberta Infants Motor Scale (AIMS)] and clinical symptoms (parental questionnaire) were recorded at entry and after 1 mo. During follow-up, cobalamin supplementation changed all markers of impaired cobalamin status (ie, plasma tHcy decreased by 54%, and MMA decreased by 84%), whereas no significant changes were seen in the placebo group (P < 0.001). The median (IQR) increase in the AIMS score was higher in the cobalamin group than in the placebo group [7.0 (5.0, 9.0) compared with 4.5 (3.3, 6.0); P = 0.003], and a higher proportion showed improvements in regurgitations (69% compared with 29%, respectively; P = 0.003). In infants with biochemical signs of impaired cobalamin function, 1 intramuscular injection of cobalamin resulted in biochemical evidence of cobalamin repletion and improvement in motor function and regurgitations, which suggest that an adequate cobalamin status is important for a rapidly developing nervous system. This trial was registered at clinicaltrials.gov as NCT00710359 and NCT00710138.

Hierarchical thinking in network biology: the unbiased modularization of biochemical networks.

PubMed

Papin, Jason A; Reed, Jennifer L; Palsson, Bernhard O

2004-12-01

As reconstructed biochemical reaction networks continue to grow in size and scope, there is a growing need to describe the functional modules within them. Such modules facilitate the study of biological processes by deconstructing complex biological networks into conceptually simple entities. The definition of network modules is often based on intuitive reasoning. As an alternative, methods are being developed for defining biochemical network modules in an unbiased fashion. These unbiased network modules are mathematically derived from the structure of the whole network under consideration.

Bivariate and multivariate analyses of the correlations between stability of the erythrocyte membrane, serum lipids and hematological variables.

PubMed

Bernardino Neto, M; de Avelar, E B; Arantes, T S; Jordão, I A; da Costa Huss, J C; de Souza, T M T; de Souza Penha, V A; da Silva, S C; de Souza, P C A; Tavares, M; Penha-Silva, N

2013-01-01

The observation that the fluidity must remain within a critical interval, outside which the stability and functionality of the cell tends to decrease, shows that stability, fluidity and function are related and that the measure of erythrocyte stability allows inferences about the fluidity or functionality of these cells. This study determined the biochemical and hematological variables that are directly or indirectly related to erythrocyte stability in a population of 71 volunteers. Data were evaluated by bivariate and multivariate analysis. The erythrocyte stability showed a greater association with hematological variables than the biochemical variables. The RDW stands out for its strong correlation with the stability of erythrocyte membrane, without being heavily influenced by other factors. Regarding the biochemical variables, the erythrocyte stability was more sensitive to LDL-C. Erythrocyte stability was significantly associated with RDW and LDL-C. Thus, the level of LDL-C is a consistent link between stability and functionality, suggesting that a measure of stability could be more one indirect parameter for assessing the risk of degenerative processes associated with high levels of LDL-C.

Effects of a 12-Week Exercise Training Program on Physical Function in Institutionalized Frail Elderly

PubMed Central

Ferreira, Cristiane Batisti; Teixeira, Pâmela dos Santos; Alves dos Santos, Geiane; Dantas Maya, Athila Teles; Americano do Brasil, Paula; Souza, Vinícius Carolino; Córdova, Cláudio; Lima, Ricardo Moreno; Nóbrega, Otávio de Toledo

2018-01-01

With the increase in life expectancy, the Brazilian elderly population has risen considerably. However, longevity is usually accompanied by problems such as the loss of functional capacity, cognitive decline, frailty syndrome, and deterioration in anthropometric parameters, particularly among those living in long-term care facilities. This randomized controlled trial aimed to verify the effects of exercise training on biochemical, inflammatory, and anthropometric indices and functional performance in institutionalized frail elderly. The sample consisted of 37 elderly people of both genders, aged 76.1 ± 7.7 years, who were randomly allocated into 2 groups: 13 individuals in the exercise group (EG) and 24 in the control group (CG). Anthropometrics, clinical history, functional tests, and biochemical evaluation were measured before and after the completion of a physical exercise program, which lasted for 12 weeks. The 12-week exercise program for frail elderly residents in a long-term care facility was efficient in improving muscle strength, speed, agility, and biochemical variables, with reversal of the frailty condition in a considerable number. However, no effects in anthropometric and inflammatory parameters were noted. PMID:29593907

Cerebellar abiotrophy in a family of Border Collie dogs.

PubMed

Sandy, J R; Slocombe, R E; Mitten, R W; Jedwab, D

2002-11-01

Cerebellar abiotrophies have a nonsex-linked, autosomal, recessively inherited basis in a number of species, and lesions typically reflect profound and progressive loss of Purkinje cells. In this report, an unusual form of abiotrophy is described for two sibling Border Collies. Extensive loss of the cerebellar granular cell layer was present with relative sparing of Purkinje cells of two female pups. The biochemical basis for this form of cerebellar abiotrophy is unknown, but the lack of disease in other siblings supports an autosomal recessive mode of inheritance.

Secondary Metabolites from Marine Microorganisms. II. Marine Fungi and Their Habitats.

PubMed

Orlova, T I; Bulgakova, V G; Polin, A N

Marine-derived fungi are of great interest as a new promising source of biologically active products such as anticancer compounds, antibiotics, inhibitors of biochemical processes. Since marine organisms inhabit biologically competitive environment with unique conditions, the chemical diversity of the secondary metabolites from marine fungi is considerably high. Recent genomic studies demonstrated that fungi can carry gene clasters encoding production of previously unknown secondary metabolites. Activation of the attenuated or silent genes would be useful either for improving activities of the known compounds or for discovery of new products.

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

USDA-ARS?s Scientific Manuscript database

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

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.

Litchi chinensis as a Functional Food and a Source of Antitumor Compounds: An Overview and a Description of Biochemical Pathways.

PubMed

Emanuele, Sonia; Lauricella, Marianna; Calvaruso, Giuseppe; D'Anneo, Antonella; Giuliano, Michela

2017-09-08

Litchi is a tasty fruit that is commercially grown for food consumption and nutritional benefits in various parts of the world. Due to its biological activities, the fruit is becoming increasingly known and deserves attention not only for its edible part, the pulp, but also for its peel and seed that contain beneficial substances with antioxidant, cancer preventive, antimicrobial, and anti-inflammatory functions. Although literature demonstrates the biological activity of Litchi components in reducing tumor cell viability in in vitro or in vivo models, data about the biochemical mechanisms responsible for these effects are quite fragmentary. This review specifically describes, in a comprehensive analysis, the antitumor properties of the different parts of Litchi and highlights the main biochemical mechanisms involved.

A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

PubMed

Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

2017-06-27

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

An extended family of novel vertebrate photopigments is widely expressed and displays a diversity of function

PubMed Central

Davies, Wayne I.L.; Tamai, T. Katherine; Zheng, Lei; Fu, Josephine K.; Rihel, Jason; Foster, Russell G.; Whitmore, David; Hankins, Mark W.

2015-01-01

Light affects animal physiology and behavior more than simply through classical visual, image-forming pathways. Nonvisual photoreception regulates numerous biological systems, including circadian entrainment, DNA repair, metabolism, and behavior. However, for the majority of these processes, the photoreceptive molecules involved are unknown. Given the diversity of photophysiological responses, the question arises whether a single photopigment or a greater diversity of proteins within the opsin superfamily detect photic stimuli. Here, a functional genomics approach identified the full complement of photopigments in a highly light-sensitive model vertebrate, the zebrafish (Danio rerio), and characterized their tissue distribution, expression levels, and biochemical properties. The results presented here reveal the presence of 42 distinct genes encoding 10 classical visual photopigments and 32 nonvisual opsins, including 10 novel opsin genes comprising four new pigment classes. Consistent with the presence of light-entrainable circadian oscillators in zebrafish, all adult tissues examined expressed two or more opsins, including several novel opsins. Spectral and electrophysiological analyses of the new opsins demonstrate that they form functional photopigments, each with unique chromophore-binding and wavelength specificities. This study has revealed a remarkable number and diversity of photopigments in zebrafish, the largest number so far discovered for any vertebrate. Found in amphibians, reptiles, birds, and all three mammalian clades, most of these genes are not restricted to teleosts. Therefore, nonvisual light detection is far more complex than initially appreciated, which has significant biological implications in understanding photoreception in vertebrates. PMID:26450929

Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja).

PubMed

Song, Yu; Zhang, Hang; You, Hongguang; Liu, Yuanming; Chen, Chao; Feng, Xu; Yu, Xingyu; Wu, Shengyang; Wang, Libo; Zhong, Shihua; Li, Qiang; Zhu, Yanming; Ding, Xiaodong

2018-04-17

The plant sucrose nonfermenting kinase 1 (SnRK1) kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth, and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library-scale screens by the means of yeast two-hybrid to identify its interacting proteins. The putative interactions were verified by yeast retransformation and β-galactosidase assays, and the selected interactions were further confirmed in planta by bimolecular fluorescence complementation and biochemical Co-IP assays. Protein phosphorylation analyses were carried out by phos-tag assay and anti-phospho-(Ser/Thr) substrate antibodies. Finally, we obtained 24 GsSnRK1 interactors and several putative substrates that can be categorized into SnRK1 regulatory β subunit, protein modification, biotic and abiotic stress-related, hormone perception and signalling, gene expression regulation, water and nitrogen transport, metabolism, and unknown proteins. Intriguingly, we first discovered that GsSnRK1 interacted with and phosphorylated the components of soybean nodulation and symbiotic nitrogen fixation. The interactions and potential functions of GsSnRK1 and its associated proteins were extensively discussed and analysed. This work provides plausible clues to elucidate the novel functions of SnRK1 in response to variable environmental, metabolic, and physiological requirements. © 2018 John Wiley & Sons Ltd.

Crystal Structure of VC0702 at 2.0 angstrom: A Conserved Hypothetical Protein from Vibrio Cholerae

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

Ni, Shuisong; Forouhar, Farhad; Bussiere, Dirksen E.

2006-06-01

VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a putative three-gene operon containing the MbaA gene, which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0? and refined to Rwork=22.8% and Rfree=26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a=66.61 ?, b=88.118 ?, and c=118.35 ? with a homodimer in the asymmetric unit. VC0702 belongs to the Pfam DUF84 and COG1986 family of proteins. Sequence conservation within the DUF84 and COG1986 families wasmore » used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeshii, which has been identified as a novel NTPase. The NTP-binding site in Mj0226 is similarly located in comparison to the conserved patch of surface residues in VC0702. Furthermore, the NTP binds to MJ0226 in a cleft and deep cavity, features that are present in the VC0702 structure as well, suggesting that VC0702 may have a biochemical function involving NTP binding that is associated with a cellular function of regulating biofilm formation in Vibrio cholerae.« less

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

PubMed

Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

2015-12-28

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

Mays, Suzanne G.; Okafor, C. Denise; Tuntland, Micheal L.

Peroxisome proliferator-activated gamma coactivator 1-α (PGC1α) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1α is known to bind and activate LRH-1, mechanisms through which PGC1α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1–PGC1α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), inmore » coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1–PGC1α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1α but promoted allosteric signaling from the helix 6/β-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1–PGC1α interaction and may illuminate strategies for selective therapeutic targeting of PGC1α-dependent LRH-1 signaling pathways.« less

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

PubMed Central

Mir, Riyaz A.; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A.; Ammons, Shalis A.; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B.; Qiu, Fang; Band, Hamid

2015-01-01

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. PMID:26711270

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

PubMed

Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2009-04-01

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

PubMed

Rajput, Nasir Ahmed; Zhang, Meixiang; Shen, Danyu; Liu, Tingli; Zhang, Qimeng; Ru, Yanyan; Sun, Peng; Dou, Daolong

2015-12-01

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

QTL mapping and molecular characterization of the classical D locus controlling seed and flower color in Linum usitatissimum (flax).

PubMed

Sudarshan, Gurudatt Pavagada; Kulkarni, Manoj; Akhov, Leonid; Ashe, Paula; Shaterian, Hamid; Cloutier, Sylvie; Rowland, Gordon; Wei, Yangdou; Selvaraj, Gopalan

2017-11-16

The flowers of flax (linseed) are blue-hued, ephemeral and self-pollinating, and the seeds are typically brown. A century-old interest in natural yellow seed variants and a historical model point to recessive alleles in B1, D and G loci being responsible, but the functional aspects had remained unknown. Here, we characterized the "D" locus by quantitative trait loci (QTL) mapping and identified a FLAVONOID 3'5' HYDROXYLASE (F3'5'H) gene therein. It does not belong to the F3'5'H clade, but resembles biochemically characterized F3'Hs (flavonoid 3' hydroxylase) but without F3'H activity. The genome lacks other F3'H or F3'H-like genes. The apparent neo-functionalization from F3'H is associated with a Thr 498  → Ser 498 substitution in a substrate recognition site (SRS). The yellow seed and white flower phenotypes of the classical d mutation was found to be due to one nucleotide deletion that would truncate the deduced product and remove three of the six potential SRS, negatively impacting delphinidin synthesis. Delphinidin is sporadic in angiosperms, and flax has no known pollination syndrome(s) with functional pollinator group(s) that are attracted to blue flowers, raising questions on the acquisition of F3'5'H. The appearance of d allele is suggestive of the beginning of the loss of F3'5'H in this species.

Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions.

PubMed

Balhana, Ricardo J C; Singla, Ashima; Sikder, Mahmudul Hasan; Withers, Mike; Kendall, Sharon L

2015-06-27

Mycobacteria inhabit diverse niches and display high metabolic versatility. They can colonise both humans and animals and are also able to survive in the environment. In order to succeed, response to environmental cues via transcriptional regulation is required. In this study we focused on the TetR family of transcriptional regulators (TFTRs) in mycobacteria. We used InterPro to classify the entire complement of transcriptional regulators in 10 mycobacterial species and these analyses showed that TFTRs are the most abundant family of regulators in all species. We identified those TFTRs that are conserved across all species analysed and those that are unique to the pathogens included in the analysis. We examined genomic contexts of 663 of the conserved TFTRs and observed that the majority of TFTRs are separated by 200 bp or less from divergently oriented genes. Analyses of divergent genes indicated that the TFTRs control diverse biochemical functions not limited to efflux pumps. TFTRs typically bind to palindromic motifs and we identified 11 highly significant novel motifs in the upstream regions of divergently oriented TFTRs. The C-terminal ligand binding domain from the TFTR complement in M. tuberculosis showed great diversity in amino acid sequence but with an overall architecture common to other TFTRs. This study suggests that mycobacteria depend on TFTRs for the transcriptional control of a number of metabolic functions yet the physiological role of the majority of these regulators remain unknown.

Characterizing the structure-function relationship reveals the mode of action of a novel antimicrobial peptide, P1, from jumper ant Myrmecia pilosula.

PubMed

Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Chinpan

2017-06-01

Microbial infections of antibiotic-resistant strains cause serious diseases and have a significant impact on public health worldwide, so novel antimicrobial drugs are urgently needed. Insect venoms, a rich source of bioactive components containing antimicrobial peptides (AMPs), are attractive candidates for new therapeutic agents against microbes. Recently, a novel peptide, P1, identified from the venom of the Australian jumper ant Myrmecia pilosula, showed potent antimicrobial activities against both Gram-negative and Gram-positive bacteria, but its structure-function relationship is unknown. Here, we used biochemical and biophysical techniques coupled with computational simulations to explore the mode of action of P1 interaction with dodecylphosphocholine (DPC) micelles as a model membrane system. Our circular dichroism (CD) and NMR studies revealed an amphipathic α-helical structure for P1 upon interaction with DPC micelles. A paramagnetic relaxation enhancement approach revealed that P1 orients its α-helix segment (F6-G14) into DPC micelles. In addition, the α-helix segment could be essential for membrane permeabilization and antimicrobial activity. Moreover, the arginine residues R8, R11, and R15 significantly contribute to helix formation and membrane-binding affinity. The lysine residue K19 of the C-terminus functionally guides P1 to interact with DPC micelles in the early interaction stage. Our study provides insights into the mode of action of P1, which is valuable in modifying and developing potent AMPs as antibiotic drugs.

Kribbella deserti sp. nov., isolated from rhizosphere soil of Ammopiptanthus mongolicus.

PubMed

Sun, Ji-Quan; Xu, Lian; Guo, Yan; Li, Wei-Le; Shao, Zhong-Qiu; Yang, Yuan-Liang; Wu, Xiao-Lei

2017-03-01

A Gram-stain-positive, aerobic bacterial strain, designated SL15-1 T , was isolated from desert soil which was sampled from the rhizosphere of Ammopiptanthus mongolicus, Hangjin Banner, Ordos, Inner Mongolia, northern China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SL15-1 T was clustered with Kribbella strains, sharing the highest similarity of 16S rRNA gene sequence (96.97 %) with Kribbella sandramycini DSM 15626 T . Strain SL15-1 T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unknown phospholipid, an unknown lipid and two unknown aminophospholipids as the major polar lipids. MK-9(H4) was the predominant menaquinone, while anteiso-C15 : 0, iso-C16 : 0, C17 : 1ω8c and iso-C14 : 0 were the major cellular fatty acids. Its genomic DNA G+C content was 65.3 mol%. The results of physiological and biochemical tests allowed the discrimination of strain SL15-1 T from its phylogenetic relatives. Kribbella deserti sp. nov. is therefore proposed with strain SL15-1 T (=CGMCC 1.15906 T =KCTC 39825 T ) as the type strain.

Canavan disease. Analysis of the nature of the metabolic lesions responsible for development of the observed clinical symptoms.

PubMed

Baslow, M H; Resnik, T R

1997-10-01

Canavan disease (CD), a rare recessive autosomal genetic disorder, is characterized by early onset and a progressive spongy degeneration of the brain involving loss of the axon's myelin sheath. After a relatively normal birth, homozygous individuals generally develop clinical symptoms within months, and usually die within several years of the onset of the disease. A biochemical defect associated with this disease results in reduced activity of the enzyme N-acetyl-L-aspartate amidohydrolase (aspartoacylase) and affected individuals have less ability to hydrolyze N-acetyl-L-asparate (NAA) in brain and other tissues. As a result of aspartoacylase deficiency, NAA builds up in extracellular fluids (ECF) and is excreted in urine. From an analysis of the NAA biochemical cycle in various tissues of many vertebrate species, evidence is presented that there may be two distinct NAA circulation patterns related to aspartoacylase activity. These include near-field circulations in the brain and the eye, and a far-field systemic circulation involving the liver and kidney, the purpose of which in each case is apparently to regenerate aspartate (Asp) in order for it to be recycled into NAA as part of the still unknown function of the NAA cycle. Based on the authors' analysis, they have also identified several metabolic outcomes of the genetic biochemical aspartoacylase lesion. First, there is a daily induced Asp deficit in the central nervous system (CNS) that is at least six times the static level of available free Asp. Second, there is up to a 50-fold drop in the intercompartmental NAA gradient, and third, the ability of the brain to perform its normal intercompartmental cycling of NAA to Asp is terminated, and as a result, the only remaining long-term source of Asp for NAA synthesis is via nutritional supplementation of Asp or its metabolic precursors. Finally, the authors identify a potential maternal-fetal interaction that may be responsible for observed normal fetal development in utero, and that provides a rationale for, and suggests how, CD might respond to far-field nutritional, transplantation, or genetic engineering techniques to alter the course of the disease.

Kinetic Competition between Elongation Rate and Binding of NELF Controls Promoter Proximal Pausing

PubMed Central

Li, Jian; Liu, Yingyun; Rhee, Ho Sung; Ghosh, Saikat Kumar B.; Bai, Lu; Pugh, B. Franklin; Gilmour, David S.

2013-01-01

Summary Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes, but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation. PMID:23746353

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

PubMed

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

Impact of 68Ga-PSMA-11 PET on Management in Patients with Biochemically Recurrent Prostate Cancer.

PubMed

Hope, Thomas A; Aggarwal, Rahul; Chee, Bryant; Tao, Dora; Greene, Kirsten L; Cooperberg, Matthew R; Feng, Felix; Chang, Albert; Ryan, Charles J; Small, Eric J; Carroll, Peter R

2017-12-01

The purpose of this prospective study was to estimate the effect of 68 Ga-labeled prostate-specific membrane antigen (PSMA)-11 PET on the intended management of patients with biochemically recurrent prostate cancer. Methods: Pre- and postimaging surveys were filled out by the referring providers for patients with biochemical recurrence who were imaged using 68 Ga-PSMA-11 PET. The inclusion criterion for this study was a prostate-specific antigen (PSA) doubling time of less than 12 mo after initial treatment (NCT02611882). Of the 150 consecutive patients imaged, 126 surveys were completed (84% response rate). The responses were categorized as major change, minor change, no change, or unknown change. Results: There were 103 patients (82%) with disease detected on 68 Ga-PSMA-11 PET. On the basis of the survey results, there were 67 patients (53.2%) with major changes in management and 8 patients (6.4%) with minor changes. The proportion of cases resulting in a change in management did not significantly differ by baseline PSA level. In patients with PSA levels below 0.2 ng/dL, 7 of 12 patients had disease detected on 68 Ga-PSMA-11 PET, 5 of whom had a major change in management. Conclusion: 68 Ga-PSMA-11 PET resulted in a major change in management in 53% of patients with biochemical recurrence. Further studies are warranted to investigate whether PSMA-based management strategies result in improved outcomes for patients. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Biochemical properties and subcellular localization of tyrosine aminotransferases in Arabidopsis thaliana.

PubMed

Wang, Minmin; Toda, Kyoko; Maeda, Hiroshi A

2016-12-01

Plants produce various L-tyrosine (Tyr)-derived compounds that are of pharmaceutical or nutritional importance to humans. Tyr aminotransferase (TAT) catalyzes the reversible transamination between Tyr and 4-hydroxyphenylpyruvate (HPP), the initial step in the biosynthesis of many Tyr-derived plant natural products. Herein reported is the biochemical characterization and subcellular localization of TAT enzymes from the model plant Arabidopsis thaliana. Phylogenetic analysis showed that Arabidopsis has at least two homologous TAT genes, At5g53970 (AtTAT1) and At5g36160 (AtTAT2). Their recombinant enzymes showed distinct biochemical properties: AtTAT1 had the highest activity towards Tyr, while AtTAT2 exhibited a broad substrate specificity for both amino and keto acid substrates. Also, AtTAT1 favored the direction of Tyr deamination to HPP, whereas AtTAT2 preferred transamination of HPP to Tyr. Subcellular localization analysis using GFP-fusion proteins and confocal microscopy showed that AtTAT1, AtTAT2, and HPP dioxygenase (HPPD), which catalyzes the subsequent step of TAT, are localized in the cytosol, unlike plastid-localized Tyr and tocopherol biosynthetic enzymes. Furthermore, subcellular fractionation indicated that, while HPPD activity is restricted to the cytosol, TAT activity is detected in both cytosolic and plastidic fractions of Arabidopsis leaf tissue, suggesting that an unknown aminotransferase(s) having TAT activity is also present in the plastids. Biochemical and cellular analyses of Arabidopsis TATs provide a fundamental basis for future in vivo studies and metabolic engineering for enhanced production of Tyr-derived phytochemicals in plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Universal dynamical properties preclude standard clustering in a large class of biochemical data.

PubMed

Gomez, Florian; Stoop, Ralph L; Stoop, Ruedi

2014-09-01

Clustering of chemical and biochemical data based on observed features is a central cognitive step in the analysis of chemical substances, in particular in combinatorial chemistry, or of complex biochemical reaction networks. Often, for reasons unknown to the researcher, this step produces disappointing results. Once the sources of the problem are known, improved clustering methods might revitalize the statistical approach of compound and reaction search and analysis. Here, we present a generic mechanism that may be at the origin of many clustering difficulties. The variety of dynamical behaviors that can be exhibited by complex biochemical reactions on variation of the system parameters are fundamental system fingerprints. In parameter space, shrimp-like or swallow-tail structures separate parameter sets that lead to stable periodic dynamical behavior from those leading to irregular behavior. We work out the genericity of this phenomenon and demonstrate novel examples for their occurrence in realistic models of biophysics. Although we elucidate the phenomenon by considering the emergence of periodicity in dependence on system parameters in a low-dimensional parameter space, the conclusions from our simple setting are shown to continue to be valid for features in a higher-dimensional feature space, as long as the feature-generating mechanism is not too extreme and the dimension of this space is not too high compared with the amount of available data. For online versions of super-paramagnetic clustering see http://stoop.ini.uzh.ch/research/clustering. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Lipogenesis in a wing-polymorphic cricket: Canalization versus morph-specific plasticity as a function of nutritional heterogeneity.

PubMed

Zera, Anthony J; Clark, Rebecca; Behmer, Spence

2016-12-01

The influence of variable nutritional input on life history adaptation is a central, but incompletely understood aspect of life history physiology. The wing-polymorphic cricket, Gryllus firmus, has been extensively studied with respect to the biochemical basis of life history adaptation, in particular, modification of lipid metabolism that underlies the enhanced accumulation of lipid flight fuel in the dispersing morph [LW(f)=long wings with functional flight muscles] relative to the flightless (SW=short-winged) morph. To date, biochemical studies have been undertaken almost exclusively using a single laboratory diet. Thus, the extent to which nutritional heterogeneity, likely experienced in the field, influences this key morph adaptation is unknown. We used the experimental approach of the Geometric Framework for Nutrition and employed 13 diets that differed in the amounts and ratios of protein and carbohydrate to assess how nutrient amount and balance affects morph-specific lipid biosynthesis. Greater lipid biosynthesis and allocation to the soma in the LW(f) compared with the SW morph (1) occurred across the entire protein-carbohydrate landscape and (2) is likely an important contributor to elevated somatic lipid in the LW(f) morph across the entire protein-carbohydrate landscape. Nevertheless, dietary carbohydrate strongly affected lipid biosynthesis in a morph-specific manner (to a greater degree in the LW(f) morph). Lipogenesis in the SW morph may be constrained due to its more limited lipid storage capacity compared to the LW(f) morph. Elevated activity of NADP + -isocitrate dehydrogenase (NADP + -IDH), an enzyme that produces reducing equivalents for lipid biosynthesis, was correlated with and may be an important cause of the increased lipogenesis in the LW(f) morph across most, but not all regions of the protein-carbohydrate landscape. By contrast, ATP-citrate lyase (ACL), an enzyme that catalyzes the first step in the pathway of fatty acid biosynthesis, showed complex morph-specific patterns of activity that were strongly contingent upon diet. Morph-specific patterns of NADP + -IDH and ACL activities across the nutrient landscape were much more complex than expected from previous studies on a single diet. Collectively, our results indicate that the biochemical basis of an important life history adaptation, morph-specific lipogenesis, can be canalized in the face of substantial nutritional heterogeneity. However, in some regions of the protein-carbohydrate landscape, it is strongly modulated in a morph-specific manner. Copyright © 2016 Elsevier Ltd. All rights reserved.

Clinical and genetic spectrum in limb-girdle muscular dystrophy type 2E.

PubMed

Semplicini, Claudio; Vissing, John; Dahlqvist, Julia R; Stojkovic, Tanya; Bello, Luca; Witting, Nanna; Duno, Morten; Leturcq, France; Bertolin, Cinzia; D'Ambrosio, Paola; Eymard, Bruno; Angelini, Corrado; Politano, Luisa; Laforêt, Pascal; Pegoraro, Elena

2015-04-28

To determine the clinical spectrum of limb-girdle muscular dystrophy 2E (LGMD2E) and to investigate whether genetic or biochemical features can predict the phenotype of the disease. All LGMD2E patients followed in participating centers were included. A specific clinical protocol was created, including quantitative evaluation of motor, respiratory, and cardiac function. Phenotype was defined as severe or mild if the age at loss of ambulation occurred before or after 18 years. Molecular analysis of SGCB gene and biochemical features of muscle biopsies were reviewed. Thirty-two patients were included (16 male, 16 female; age 7-67 years; 15 severe, 12 mild, and 5 unknown). Neurologic examination showed proximal muscle weakness in all patients, but distal involvement was also observed in patients with severe disease early in the disease course. Cardiac involvement was observed in 20 patients (63%) even before overt muscle involvement. Six patients had restrictive respiratory insufficiency requiring assisted ventilation (19%). Seventeen different mutations were identified, and 3 were recurrent. The c.377_384dup (13 alleles) was associated with the severe form, the c.-22_10dup (10) with the milder form, and the c.341C>T (9) with both. The entire sarcoglycan complex was undetectable by muscle immunohistochemistry or Western blot in 9/10 severe cases and reduced in 7/7 mild cases. The residual amount of sarcoglycan in muscle resulted a predictor of age at loss of ambulation. This study expands the spectrum of phenotype in β-sarcoglycanopathy and provides strong evidence that severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan protein expression. © 2015 American Academy of Neurology.

Clinical and genetic spectrum in limb-girdle muscular dystrophy type 2E

PubMed Central

Semplicini, Claudio; Vissing, John; Dahlqvist, Julia R.; Stojkovic, Tanya; Bello, Luca; Witting, Nanna; Duno, Morten; Leturcq, France; Bertolin, Cinzia; D'Ambrosio, Paola; Eymard, Bruno; Angelini, Corrado; Politano, Luisa

2015-01-01

Objective: To determine the clinical spectrum of limb-girdle muscular dystrophy 2E (LGMD2E) and to investigate whether genetic or biochemical features can predict the phenotype of the disease. Methods: All LGMD2E patients followed in participating centers were included. A specific clinical protocol was created, including quantitative evaluation of motor, respiratory, and cardiac function. Phenotype was defined as severe or mild if the age at loss of ambulation occurred before or after 18 years. Molecular analysis of SGCB gene and biochemical features of muscle biopsies were reviewed. Results: Thirty-two patients were included (16 male, 16 female; age 7–67 years; 15 severe, 12 mild, and 5 unknown). Neurologic examination showed proximal muscle weakness in all patients, but distal involvement was also observed in patients with severe disease early in the disease course. Cardiac involvement was observed in 20 patients (63%) even before overt muscle involvement. Six patients had restrictive respiratory insufficiency requiring assisted ventilation (19%). Seventeen different mutations were identified, and 3 were recurrent. The c.377_384dup (13 alleles) was associated with the severe form, the c.-22_10dup (10) with the milder form, and the c.341C>T (9) with both. The entire sarcoglycan complex was undetectable by muscle immunohistochemistry or Western blot in 9/10 severe cases and reduced in 7/7 mild cases. The residual amount of sarcoglycan in muscle resulted a predictor of age at loss of ambulation. Conclusions: This study expands the spectrum of phenotype in β-sarcoglycanopathy and provides strong evidence that severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan protein expression. PMID:25862795

Adult Onset Leigh Syndrome in the Intensive Care Setting: A Novel Presentation of a C12orf65 Related Mitochondrial Disease.

PubMed

Wesolowska, Maria; Gorman, Grainne S; Alston, Charlotte L; Pajak, Aleksandra; Pyle, Angela; He, Langping; Griffin, Helen; Chinnery, Patrick F; Miller, James A L; Schaefer, Andrew M; Taylor, Robert W; Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M

2015-10-07

Mitochondrial disease can present at any age, with dysfunction in almost any tissue making diagnosis a challenge. It can result from inherited or sporadic mutations in either the mitochondrial or the nuclear genome, many of which affect intraorganellar gene expression. The estimated prevalence of 1/4300 indicates these to be amongst the commonest inherited neuromuscular disorders, emphasising the importance of recognition of the diagnostic clinical features. Despite major advances in our understanding of the molecular basis of mitochondrial diseases, accurate and early diagnoses are critically dependent on the fastidious clinical and biochemical characterisation of patients. Here we describe a patient harbouring a previously reported homozygous mutation in C12orf65, a mitochondrial protein of unknown function, which does not adhere to the proposed distinct genotype-phenotype relationship. We performed clinical, biochemical and molecular analysis including whole exome sequencing on patient samples and cell lines. We report an extremely rare case of an adult presenting with Leigh-like disease, in intensive care, in the 5th decade of life, harbouring a recessively inherited mutation previously reported in children. A global reduction in intra-mitochondrial protein synthesis was observed despite normal or elevated levels of mt-RNA, leading to an isolated complex IV deficiency. All the reported C12orf65 mutations have shown an autosomal recessive pattern of inheritance. Mitochondrial disease causing mutations inherited in this manner are usually of early onset and associated with a severe, often fatal clinical phenotype. Presentations in adulthood are usually less severe. This patient's late adulthood presentation is in sharp contrast emphasising the clinical variability that is characteristic of mitochondrial disease and illustrates why making a definitive diagnosis remains a formidable challenge.

Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain composition of rat gastrocnemius muscles.

PubMed

Hong, Bin; Chen, Min; Hu, Xing-yue

2013-11-01

Botulinum toxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of a synaptosomal-associated protein of 25 kDa (SNAP-25). It has gained widespread use as a treatment for cerebral palsy and skeletal muscle hypertrophy. In China, Chinese botulinum toxin type A (CBTX-A), a type of BoNT/A, is in widespread clinical use. However, the changes in the morphological and biochemical properties of treated muscles and in remote muscles from the CBTX-A injection site are relatively unknown. Therefore, we investigated the changes in histomorphology and myosin heavy chain (MyHC) isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A. The weakness of the injected muscles was assessed periodically to identify their functional deficiency. Muscle slices were stained with hematoxylin-eosin (HE) and adenosine triphosphatase (ATPase). MyHC isoform composition was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to uncover changes in morphological and biochemical properties. Our findings demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles, shifts in MyHC isoform composition emerged on the third day after injection and peaked in the fourth week. The composition remained distinctly different from that of the control group after the twelfth week. More specifically, there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx, type IIa, and type I isoforms. Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that the MyHC composition remained far from normal six months after a single injection. However, no noticeable remote muscle weakness was induced.

The exercise-induced biochemical milieu enhances collagen content and tensile strength of engineered ligaments.

PubMed

West, Daniel W D; Lee-Barthel, Ann; McIntyre, Todd; Shamim, Baubak; Lee, Cassandra A; Baar, Keith

2015-10-15

Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post-exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue-engineered ligaments is enhanced by serum obtained post-exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ∼7-fold greater than resting serum (P < 0.001), whereas IGF-1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post-exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties - maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF-1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post-exercise serum activated mTORC1 and ERK1/2. In conclusion, the post-exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Structure of Thermotoga maritima Stationary Phase Survival Protein SurE: A Novel Acid Phosphatase

PubMed Central

Zhang, R.-G.; Skarina, T.; Katz, J.E.; Beasley, S.; Khachatryan, A.; Vyas, S.; Arrowsmith, C.H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.

2009-01-01

Summary Background The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase σ subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results The structure of SurE from Thermotoga maritima was determined at 2.0 Å. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5–6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and inter-subunit salt bridges were identified that may explain the SurE thermostability. Conclusions The structure of SurE provided information about the protein’s fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis. PMID:11709173

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

PubMed

Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

Prevalence of functional disorders of androgen excess in unselected premenopausal women: a study in blood donors.

PubMed

Sanchón, Raúl; Gambineri, Alessandra; Alpañés, Macarena; Martínez-García, M Ángeles; Pasquali, Renato; Escobar-Morreale, Héctor F

2012-04-01

The polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. On the contrary, the prevalences of other disorders of androgen excess such as idiopathic hyperandrogenism and idiopathic hirsutism remain unknown. We aimed to obtain an unbiased estimate of the prevalence in premenopausal women of (i) signs of androgen excess and (ii) PCOS, idiopathic hyperandrogenism and idiopathic hirsutism. A multicenter prevalence survey included 592 consecutive premenopausal women (393 from Madrid, Spain and 199 from Bologna, Italy) reporting spontaneously for blood donation. Immediately before donation, we conducted clinical and biochemical phenotyping for androgen excess disorders. We determined the prevalence of (i) hirsutism, acne and alopecia as clinical signs of androgen excess and (ii) functional disorders of androgen excess, including PCOS, defined by the National Institute of Child Health and Human Development/National Institute of Health criteria, idiopathic hyperandrogenism and idiopathic hirsutism. Regarding clinical signs of hyperandrogenism, hirsutism and acne were equally frequent [12.2% prevalence; 95% confidence interval (CI): 9.5-14.8%], whereas alopecia was uncommon (1.7% prevalence, 95% CI: 0.7-2.7%). Regarding functional disorders of androgen excess, PCOS and idiopathic hirsutism were equally frequent (5.4% prevalence, 95% CI: 3.6-7.2) followed by idiopathic hyperandrogenism (3.9% prevalence, 95% CI: 2.3-5.4). Clinical signs of hyperandrogenism and functional disorders of androgen excess show a high prevalence in premenopausal women. The prevalences of idiopathic hyperandrogenism and idiopathic hirsutism are similar to that of PCOS, highlighting the need for further research on the pathophysiology, consequences for health and clinical implications of these functional forms of androgen excess.

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana*

PubMed Central

Kotzsch, Alexander; Pawolski, Damian; Milentyev, Alexander; Shevchenko, Anna; Scheffel, André; Poulsen, Nicole; Shevchenko, Andrej; Kröger, Nils

2016-01-01

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis. PMID:26710847

Network Reconstruction Using Nonparametric Additive ODE Models

PubMed Central

Henderson, James; Michailidis, George

2014-01-01

Network representations of biological systems are widespread and reconstructing unknown networks from data is a focal problem for computational biologists. For example, the series of biochemical reactions in a metabolic pathway can be represented as a network, with nodes corresponding to metabolites and edges linking reactants to products. In a different context, regulatory relationships among genes are commonly represented as directed networks with edges pointing from influential genes to their targets. Reconstructing such networks from data is a challenging problem receiving much attention in the literature. There is a particular need for approaches tailored to time-series data and not reliant on direct intervention experiments, as the former are often more readily available. In this paper, we introduce an approach to reconstructing directed networks based on dynamic systems models. Our approach generalizes commonly used ODE models based on linear or nonlinear dynamics by extending the functional class for the functions involved from parametric to nonparametric models. Concomitantly we limit the complexity by imposing an additive structure on the estimated slope functions. Thus the submodel associated with each node is a sum of univariate functions. These univariate component functions form the basis for a novel coupling metric that we define in order to quantify the strength of proposed relationships and hence rank potential edges. We show the utility of the method by reconstructing networks using simulated data from computational models for the glycolytic pathway of Lactocaccus Lactis and a gene network regulating the pluripotency of mouse embryonic stem cells. For purposes of comparison, we also assess reconstruction performance using gene networks from the DREAM challenges. We compare our method to those that similarly rely on dynamic systems models and use the results to attempt to disentangle the distinct roles of linearity, sparsity, and derivative estimation. PMID:24732037

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum.

PubMed

Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

2018-04-01

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A 2A receptor (A 2A R) and cannabinoid CB 1 receptor (CB 1 R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A 2A R and CB 1 R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A 2A R-CB 1 R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A 2A R-CB 1 R heteromers in the dorsal striatum. Specifically, our data unveil that the A 2A R-CB 1 R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

PubMed Central

Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

2018-01-01

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases. PMID:28102227

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

PubMed

Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John

2006-01-01

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)

PubMed Central

Bayfield, Mark A.; Yang, Ruiqing; Maraia, Richard J.

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3’OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3’OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3’OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNA assembly by hLARP7/PIP7S). Analyses of other LARP family members (i.e., hLARP4, hLARP6) suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs. PMID:20138158

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs).

PubMed

Bayfield, Mark A; Yang, Ruiqing; Maraia, Richard J

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Misdiagnosis of Thyroid Disorders in Down Syndrome: Time to Re-Examine the Myth?

ERIC Educational Resources Information Center

Prasher, V.; Haque, M. S.

2005-01-01

There is a reported association between thyroid disorders and Down syndrome, but is this association based on valid and reliable research evidence? We evaluated thyroid function test results of 110 healthy adults with Down syndrome to determine biochemical thyroid status. Approximately two thirds were biochemically euthyroid when assessed by…

A master equation and moment approach for biochemical systems with creation-time-dependent bimolecular rate functions

PubMed Central

Chevalier, Michael W.; El-Samad, Hana

2014-01-01

Noise and stochasticity are fundamental to biology and derive from the very nature of biochemical reactions where thermal motion of molecules translates into randomness in the sequence and timing of reactions. This randomness leads to cell-to-cell variability even in clonal populations. Stochastic biochemical networks have been traditionally modeled as continuous-time discrete-state Markov processes whose probability density functions evolve according to a chemical master equation (CME). In diffusion reaction systems on membranes, the Markov formalism, which assumes constant reaction propensities is not directly appropriate. This is because the instantaneous propensity for a diffusion reaction to occur depends on the creation times of the molecules involved. In this work, we develop a chemical master equation for systems of this type. While this new CME is computationally intractable, we make rational dimensional reductions to form an approximate equation, whose moments are also derived and are shown to yield efficient, accurate results. This new framework forms a more general approach than the Markov CME and expands upon the realm of possible stochastic biochemical systems that can be efficiently modeled. PMID:25481130

A master equation and moment approach for biochemical systems with creation-time-dependent bimolecular rate functions

NASA Astrophysics Data System (ADS)

Chevalier, Michael W.; El-Samad, Hana

2014-12-01

Noise and stochasticity are fundamental to biology and derive from the very nature of biochemical reactions where thermal motion of molecules translates into randomness in the sequence and timing of reactions. This randomness leads to cell-to-cell variability even in clonal populations. Stochastic biochemical networks have been traditionally modeled as continuous-time discrete-state Markov processes whose probability density functions evolve according to a chemical master equation (CME). In diffusion reaction systems on membranes, the Markov formalism, which assumes constant reaction propensities is not directly appropriate. This is because the instantaneous propensity for a diffusion reaction to occur depends on the creation times of the molecules involved. In this work, we develop a chemical master equation for systems of this type. While this new CME is computationally intractable, we make rational dimensional reductions to form an approximate equation, whose moments are also derived and are shown to yield efficient, accurate results. This new framework forms a more general approach than the Markov CME and expands upon the realm of possible stochastic biochemical systems that can be efficiently modeled.

Minimal-Approximation-Based Decentralized Backstepping Control of Interconnected Time-Delay Systems.

PubMed

Choi, Yun Ho; Yoo, Sung Jin

2016-12-01

A decentralized adaptive backstepping control design using minimal function approximators is proposed for nonlinear large-scale systems with unknown unmatched time-varying delayed interactions and unknown backlash-like hysteresis nonlinearities. Compared with existing decentralized backstepping methods, the contribution of this paper is to design a simple local control law for each subsystem, consisting of an actual control with one adaptive function approximator, without requiring the use of multiple function approximators and regardless of the order of each subsystem. The virtual controllers for each subsystem are used as intermediate signals for designing a local actual control at the last step. For each subsystem, a lumped unknown function including the unknown nonlinear terms and the hysteresis nonlinearities is derived at the last step and is estimated by one function approximator. Thus, the proposed approach only uses one function approximator to implement each local controller, while existing decentralized backstepping control methods require the number of function approximators equal to the order of each subsystem and a calculation of virtual controllers to implement each local actual controller. The stability of the total controlled closed-loop system is analyzed using the Lyapunov stability theorem.

Nanoparticles as biochemical sensors

PubMed Central

El-Ansary, Afaf; Faddah, Layla M

2010-01-01

There is little doubt that nanoparticles offer real and new opportunities in many fields, such as biomedicine and materials science. Such particles are small enough to enter almost all areas of the body, including cells and organelles, potentially leading to new approaches in nanomedicine. Sensors for small molecules of biochemical interest are of critical importance. This review is an attempt to trace the use of nanomaterials in biochemical sensor design. The possibility of using nanoparticles functionalized with antibodies as markers for proteins will be elucidated. Moreover, capabilities and applications for nanoparticles based on gold, silver, magnetic, and semiconductor materials (quantum dots), used in optical (absorbance, luminescence, surface enhanced Raman spectroscopy, surface plasmon resonance), electrochemical, and mass-sensitive sensors will be highlighted. The unique ability of nanosensors to improve the analysis of biochemical fluids is discussed either through considering the use of nanoparticles for in vitro molecular diagnosis, or in the biological/biochemical analysis for in vivo interaction with the human body. PMID:24198472

Beyond the antioxidant: the double life of vitamin C.

PubMed

De Tullio, Mario C

2012-01-01

When considering the history of vitamin C, and the names given to this molecule in early days, the Latin proverb nomen est omen suddenly comes to mind. Around 1920, when Casimir Funk introduced the term Vitamin C to indicate the nutritional factor necessary to prevent the pathological state known as scurvy, the nature of the active molecule was still unknown (Davies MB, Austin J, Partridge DA (1991) Vitamin C: Its chemistry and biochemistry. The Royal Society of Chemistry, Cambridge UK). Almost in the same years, Albert Szent-Giörgyi was striving to identify a new 6-carbon sugar he had obtained in crystal form from oranges, lemons, cabbage and adrenal glands. As humorously described by Szent-Giörgyi himself (Szent-Giörgyi A (1963) Lost in the twentieth century. Annu Rev Biochem 36:1-15), he intended to name this yet unknown carbohydrate "ignose". When this name was rejected by Sir Arthur Harden, editor of the Biochemical Journal, he suggested to name it "godnose", meaning that only God could know the real identity of the molecule. Obviously, also this choice was considered inappropriate by Harden, who suggested the plain name "hexuronic acid". Only later, when the structure of "hexuronic acid" had been completely elucidated, and biological tests performed by Swirbely identified this molecule as the anti-scurvy factor vitamin C, Szent-Giörgyi and Walter Norman Haworth decided to eventually name it ascorbic acid (Szent-Giörgyi A (1963) Lost in the twentieth century. Annu Rev Biochem 36:1-15). "Ascorbic" literally means "against scurvy", and scurvy is known to be mainly due to the inactivation of some important dioxygenases involved in the synthesis of a few key molecules, including different collagen forms (De Tullio MC (2004) How does ascorbic acid prevent scurvy? A survey of the nonantioxidant functions of vitamin C. In: Asard H, May J, Smirnoff N (eds) Vitamin C, its functions and biochemistry in animals and plants. Bios Scientific Publishers, Oxford, UK, pp. 159-172). All this has very little to do with the celebrated role of ascorbic acid (ASC) as an antioxidant. So, if the fate of ASC had to be found in its name, its role in the prevention of scurvy (i.e. beyond the antioxidant function) should be considered its main feature. But, in spite of more than 80 years of extensive research (34,424 hits in a PubMed query on January 6 2007), an unprecedented popularity among the general public, an estimated market of several billion dollars (Hancock RD, Viola R (2005) Improving the nutritional value of crops through enhancement of l-ascorbic acid (vitamin C) content: Rationale and biotechnological opportunities. J Agr Food Chem 53:5248-5257), we should honestly conclude that the fate of vitamin C is still in the first name it received, many years ago: we still ignore much of its actual relevance in cell metabolism, although we are progressively getting aware of the many facets of this fascinating molecule, and its direct involvement in the regulation of apparently unrelated pathways (Arrigoni O, De Tullio MC (2002) Ascorbic acid, much more than just an antioxidant. Biochim Biophys Acta 1569:1-9; De Tullio MC, Arrigoni O (2004) Hopes, disillusions and more hopes from vitamin C. Cell Mol Life Sci 61:209-219; Duarte TL, Lunec J (2005) When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Rad Res 39:671-686). Recent data on ASC involvement in cell signalling and gene expression open new perspectives, that will be presented and discussed in this chapter.

Lessons from (patho)physiological tissue stiffness and their implications for drug screening, drug delivery and regenerative medicine.

PubMed

Chen, Wen Li Kelly; Simmons, Craig A

2011-04-30

Diseased tissues are noted for their compromised mechanical properties, which contribute to organ failure; regeneration entails restoration of tissue structure and thereby functions. Thus, the physical signature of a tissue is closely associated with its biological function. In this review, we consider a mechanics-centric view of disease and regeneration by drawing parallels between in vivo tissue-level observations and corroborative cellular evidence in vitro to demonstrate the importance of the mechanical stiffness of the extracellular matrix in these processes. This is not intended to devalue the importance of biochemical signaling; in fact, as we discuss, many mechanical stiffness-driven processes not only require cooperation with biochemical cues, but they ultimately converge at common signaling cascades to influence cell and tissue function in an integrative manner. The study of how physical and biochemical signals collectively modulate cell function not only brings forth a more holistic understanding of cell (patho)biology, but it also creates opportunities to control material properties to improve culture platforms for research and drug screening and aid in the rationale design of biomaterials for molecular therapy and tissue engineering applications. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Early histological and functional effects of chronic copper exposure in rat liver.

PubMed

Cisternas, Felipe A; Tapia, Gladys; Arredondo, Miguel; Cartier-Ugarte, Denise; Romanque, Pamela; Sierralta, Walter D; Vial, María T; Videla, Luis A; Araya, Magdalena

2005-10-01

Cu is an essential trace element capable of producing toxic effects in animals and man when ingested acutely or chronically in excess. Although chronic Cu exposure is increasingly recognized as a public health issue, its early effects remain largely unknown. We approached the significance of a moderate chronic Cu load in young rats to correlate early hepatic histopathological changes with functional alterations of liver cells. For this purpose, supplementation with 1,200 ppm of Cu in rat food for 16 weeks was chosen. In these conditions, Cu load elicited a significant decrease in growth curves. There were mild light microscopy alterations in Cu-treated rats, although increasing intracellular Cu storage was correlated with longer Cu exposure both by histological and biochemical measurements. Ultrastructural alterations included lysosomal inclusions as well as mitochondrial and nuclear changes. Liver perfusion studies revealed higher rates of basal O(2) consumption and colloidal carbon-induced O(2) uptake in Cu-treated rats, with enhanced carbon-induced O(2)/carbon uptake ratios and NF-kappaB DNA binding activity. These changes were time-dependent and returned to control values after 12 or 16 weeks. It is concluded that subchronic Cu loading in young rats induces early hepatic morphological changes, with enhancement in Küpffer cell-dependent respiratory burst activity and NF-kappaB DNA binding, cellular responses that may prevent or alleviate the hepatotoxicity of the metal.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

PubMed

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Sall1 Maintains Nephron Progenitors and Nascent Nephrons by Acting as Both an Activator and a Repressor

PubMed Central

Kanda, Shoichiro; Tanigawa, Shunsuke; Ohmori, Tomoko; Taguchi, Atsuhiro; Kudo, Kuniko; Suzuki, Yutaka; Sato, Yuki; Hino, Shinjiro; Sander, Maike; Perantoni, Alan O.; Sugano, Sumio; Nakao, Mitsuyoshi

2014-01-01

The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling-mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown. Here, we report that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons in mice. Analysis of mice with an inducible Sall1 deletion revealed that Sall1 activates genes expressed in progenitors while repressing genes expressed in differentiating nephrons. Sall1 and Six2 co-occupied many progenitor-related gene loci, and Sall1 bound to Six2 biochemically. In contrast, Sall1 did not bind to the Wnt4 locus suppressed by Six2. Sall1-mediated repression was also independent of its binding to DNA. Thus, Sall1 maintains nephron progenitors and their derivatives by a unique mechanism, which partly overlaps but is distinct from that of Six2: Sall1 activates progenitor-related genes in Six2-positive nephron progenitors and represses gene expression in Six2-negative differentiating nascent nephrons. PMID:24744442

The antihypertensive effect of calorie restriction in obese adolescents: dissociation of effects on erythrocyte countertransport and cotransport.

PubMed

Weder, A B; Torretti, B A; Katch, V L; Rocchini, A P

1984-10-01

Measures of maximal rates of lithium-sodium countertransport and frusemide-sensitive sodium and potassium cotransport have been proposed as biochemical markers for human essential hypertension. The stability of these functions over time within the same individuals has led to the suggestion that maximal transport capacities are genetically determined. The present study confirms the reproducibility of functional assays of countertransport and cotransport in human erythrocytes after overnight storage and over a six-month period in normal volunteers and provides estimates of the magnitude of technical error for each assay. A long-term dietary intervention study in a group of obese adolescents demonstrated marked increases in erythrocyte sodium levels and maximal frusemide-sensitive sodium and potassium fluxes but no changes in cell potassium or water and no effect on lithium-sodium countertransport. A correlation between the decrease in percentage of body fat and the increase in cell sodium content suggests a link between the metabolic effects of dieting and control of erythrocyte cation handling. Although the mechanism linking dietary calorie restriction and changes in erythrocyte cation metabolism is unknown, evaluation of body weight, and especially recent weight loss, is important in studies of erythrocyte transport. Conclusions regarding genetic contributions to the activities of lithium-sodium countertransport and sodium-potassium cotransport systems will be strengthened by clarification of environmental regulators.

Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

PubMed Central

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

2011-01-01

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins. PMID:21712243

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

PubMed Central

Garg, Abhishek D; Krysko, Dmitri V; Verfaillie, Tom; Kaczmarek, Agnieszka; Ferreira, Gabriela B; Marysael, Thierry; Rubio, Noemi; Firczuk, Malgorzata; Mathieu, Chantal; Roebroek, Anton J M; Annaert, Wim; Golab, Jakub; de Witte, Peter; Vandenabeele, Peter; Agostinis, Patrizia

2012-01-01

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC-IIhigh) and functional stimulation (NOhigh, IL-10absent, IL-1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress. PMID:22252128

The Circadian Oscillator of the Cerebral Cortex: Molecular, Biochemical and Behavioral Effects of Deleting the Arntl Clock Gene in Cortical Neurons.

PubMed

Bering, Tenna; Carstensen, Mikkel Bloss; Wörtwein, Gitta; Weikop, Pia; Rath, Martin Fredensborg

2018-02-01

A molecular circadian oscillator resides in neurons of the cerebral cortex, but its role is unknown. Using the Cre-LoxP method, we have here abolished the core clock gene Arntl in those neurons. This mouse represents the first model carrying a deletion of a circadian clock component specifically in an extrahypothalamic cell type of the brain. Molecular analyses of clock gene expression in the cerebral cortex of the Arntl conditional knockout mouse revealed disrupted circadian expression profiles, whereas clock gene expression in the suprachiasmatic nucleus was still rhythmic, thus showing that Arntl is required for normal function of the cortical circadian oscillator. Daily rhythms in running activity and temperature were not influenced, whereas the resynchronization response to experimental jet-lag exhibited minor though significant differences between genotypes. The tail-suspension test revealed significantly prolonged immobility periods in the knockout mouse indicative of a depressive-like behavioral state. This phenotype was accompanied by reduced norepinephrine levels in the cerebral cortex. Our data show that Arntl is required for normal cortical clock function and further give reason to suspect that the circadian oscillator of the cerebral cortex is involved in regulating both circadian biology and mood-related behavior and biochemistry. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events.

PubMed

Herasse, M; Ono, Y; Fougerousse, F; Kimura, E; Stockholm, D; Beley, C; Montarras, D; Pinset, C; Sorimachi, H; Suzuki, K; Beckmann, J S; Richard, I

1999-06-01

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

Single-side renal sympathetic denervation to treat malignant refractory hypertension in a solitary kidney patient.

PubMed

Ribichini, Flavio; Ferrara, Angela; Pighi, Michele; Pesarini, Gabriele; Gambaro, Alessia; Valvo, Enrico; Lupo, Antonio; Vassanelli, Corrado

2014-12-01

Renal sympathetic denervation (RSD) is emerging as a new therapeutic option for patients with severe hypertension refractory to medical therapy. Patients affected by renovascular or anatomical abnormalities have hitherto been systematically excluded from clinical trials with RSD because of concern about safety and the unknown efficacy of the procedure in this subgroup of patients. We describe the management of a case of RSD in a single-kidney patient with refractory hypertension; the patient had had a previous surgical right nephrectomy for renal cell carcinoma that subsequently required no other oncologic treatment. After multidisciplinary assessment, the patient underwent RSD. The procedure was performed through a 6F femoral access using the Symplicity™ RSD system (Medtronic, Mountain View, CA, USA). Radiofrequency was applied to the renal artery wall in 6 different points under general sedation with midazolam to control back pain caused by the procedure, that was performed without periprocedural complications. The patient was discharged 2 days later after a control of the vascular access site and routine biochemical examinations. The following 9-month follow up showed a significant reduction in blood pressure and stable renal function, without signs of renal damage. Our report confirms the feasibility of RSD in this delicate context, without evident negative effects on kidney function and with a significant reduction in blood pressure. Future studies are needed to fully clarify the value of RSD in single-kidney patients.

Deviation of the typical AAA substrate-threading pore prevents fatal protein degradation in yeast Cdc48.

PubMed

Esaki, Masatoshi; Islam, Md Tanvir; Tani, Naoki; Ogura, Teru

2017-07-14

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

PubMed Central

Gordon, Catherine A.; Hartono, Stella R.; Chédin, Frédéric

2013-01-01

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease. PMID:23894490

MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

PubMed

Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

2013-03-01

For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

Novel Functions of EZH2 in Triple-Negative Breast Cancer: Translation into New Biomarker and Treatment Strategies

DTIC Science & Technology

2016-07-01

p38in both the nucleus and the cytoplasm of MDA-MB-231 cells. We will continue with biochemical and biophysical studies towards mapping the binding...descriptions of key accomplishments according to the approved statement of work for Year 1. Aim 2: Biochemical and biophysical characterization of the

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.

Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated through distinct biochemical mechanisms

PubMed Central

Huang, Chuanxin; Hatzi, Katerina; Melnick, Ari

2013-01-01

The transcription factor Bcl-6 orchestrates the germinal center reaction through its actions in B and T cells, and regulates inflammatory signaling in macrophages. We report that genetic replacement by mutant Bcl-6, which cannot bind corepressors to its BTB domain, disrupted germinal center formation and immunoglobulin affinity maturation, due to a defect in B cell proliferation and survival. In contrast, BTB loss of function had no effect on T follicular helper cell differentiation and function, nor other T helper subsets. Bcl6 null mice displayed a lethal inflammatory phenotype, whereas BTB mutant mice experienced normal healthy lives with no inflammation. Bcl-6 repression of inflammatory responses in macrophages was accordingly independent of the BTB domain repressor function. Bcl-6 thus mediates its actions through lineage-specific biochemical functions. PMID:23455674

Ferroptosis-inducing agents compromise in vitro human islet viability and function.

PubMed

Bruni, Antonio; Pepper, Andrew R; Pawlick, Rena L; Gala-Lopez, Boris; Gamble, Anissa F; Kin, Tatsuya; Seeberger, Karen; Korbutt, Gregory S; Bornstein, Stefan R; Linkermann, Andreas; Shapiro, A M James

2018-05-22

Human islet transplantation has been hampered by donor cell death associated with the islet preparation procedure before transplantation. Regulated necrosis pathways are biochemically and morphologically distinct from apoptosis. Recently, ferroptosis was identified as a non-apoptotic form of iron-dependent regulated necrosis implicated in various pathological conditions. Mediators of islet oxidative stress, including glutathione peroxidase-4 (GPX4), have been identified as inhibitors of ferroptosis, and mechanisms that affect GPX4 function can impact islet function and viability. Ferroptosis has not been investigated directly in human islets, and its relevance in islet transplantation remains unknown. Herein, we sought to determine whether in vitro human islet viability and function is compromised in the presence of two distinct ferroptosis-inducing agents (FIA), erastin or RSL3, and whether these effects could be rescued with ferroptosis inhibitors, ferrostatin-1 (Fer-1), or desferrioxamine (DFO). Viability, as assessed by lactate dehydrogenase (LDH) release, revealed significant death in erastin- and RSL3-treated islets, 20.3% ± 3.8 and 24.4% ± 2.5, 24 h post culture, respectively. These effects were ameliorated in islets pre-treated with Fer-1 or the iron chelator, desferrioxamine (DFO). Stimulation index, a marker of islet function revealed a significant reduction in function in erastin-treated islets (control 1.97 ± 0.13 vs. 50 μM erastin 1.32 ± 0.1) (p < 0.05). Fer-1 and DFO pre-treatment alone did not augment islet viability or function. Pre-treatment of islets with erastin or Fer-1 did not impact in vivo engraftment in an immunodeficient mouse transplant model. Our data reveal that islets are indeed susceptible to ferroptosis in vitro, and induction of this novel cell death modality leads to compromised islet function, which can be recoverable in the presence of the ferroptosis inhibitors. The in vivo impact of this pathway in islet transplantation remains elusive given the constraints of our study, but warrants continued investigation.

Exploring the evolution of protein function in Archaea.

PubMed

Goncearenco, Alexander; Berezovsky, Igor N

2012-05-30

Despite recent progress in studies of the evolution of protein function, the questions what were the first functional protein domains and what were their basic building blocks remain unresolved. Previously, we introduced the concept of elementary functional loops (EFLs), which are the functional units of enzymes that provide elementary reactions in biochemical transformations. They are presumably descendants of primordial catalytic peptides. We analyzed distant evolutionary connections between protein functions in Archaea based on the EFLs comprising them. We show examples of the involvement of EFLs in new functional domains, as well as reutilization of EFLs and functional domains in building multidomain structures and protein complexes. Our analysis of the archaeal superkingdom yields the dominating mechanisms in different periods of protein evolution, which resulted in several levels of the organization of biochemical function. First, functional domains emerged as combinations of prebiotic peptides with the very basic functions, such as nucleotide/phosphate and metal cofactor binding. Second, domain recombination brought to the evolutionary scene the multidomain proteins and complexes. Later, reutilization and de novo design of functional domains and elementary functional loops complemented evolution of protein function.

Recommendations for terminology and databases for biochemical thermodynamics.

PubMed

Alberty, Robert A; Cornish-Bowden, Athel; Goldberg, Robert N; Hammes, Gordon G; Tipton, Keith; Westerhoff, Hans V

2011-05-01

Chemical equations are normally written in terms of specific ionic and elemental species and balance atoms of elements and electric charge. However, in a biochemical context it is usually better to write them with ionic reactants expressed as totals of species in equilibrium with each other. This implies that atoms of elements assumed to be at fixed concentrations, such as hydrogen at a specified pH, should not be balanced in a biochemical equation used for thermodynamic analysis. However, both kinds of equations are needed in biochemistry. The apparent equilibrium constant K' for a biochemical reaction is written in terms of such sums of species and can be used to calculate standard transformed Gibbs energies of reaction Δ(r)G'°. This property for a biochemical reaction can be calculated from the standard transformed Gibbs energies of formation Δ(f)G(i)'° of reactants, which can be calculated from the standard Gibbs energies of formation of species Δ(f)G(j)° and measured apparent equilibrium constants of enzyme-catalyzed reactions. Tables of Δ(r)G'° of reactions and Δ(f)G(i)'° of reactants as functions of pH and temperature are available on the web, as are functions for calculating these properties. Biochemical thermodynamics is also important in enzyme kinetics because apparent equilibrium constant K' can be calculated from experimentally determined kinetic parameters when initial velocities have been determined for both forward and reverse reactions. Specific recommendations are made for reporting experimental results in the literature. Copyright © 2011 Elsevier B.V. All rights reserved.

Improved biochemical preservation of lung slices during cold storage.

PubMed

Bull, D A; Connors, R C; Reid, B B; Albanil, A; Stringham, J C; Karwande, S V

2000-05-15

Development of lung preservation solutions typically requires whole-organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that lung slices could be used to assess preservation of biochemical function during cold storage. Whole rat lungs were precision cut into slices with a thickness of 500 microm and preserved at 4 degrees C in the following solutions: University of Wisconsin (UW), Euro-Collins (EC), low-potassium-dextran (LPD), Kyoto (K), normal saline (NS), or a novel lung preservation solution (NPS) developed using this model. Lung biochemical function was assessed by ATP content (etamol ATP/mg wet wt) and capacity for protein synthesis (cpm/mg protein) immediately following slicing (0 h) and at 6, 12, 18, and 24 h of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as means +/- SD. ATP content was significantly higher in the lung slices stored in NPS compared with all other solutions at each time point (P < 0.0001). Protein synthesis was significantly higher in the lung slices stored in NPS compared with all other solutions at 6, 12, and 18 h of preservation (P < 0.05). This lung slice model allows the rapid and efficient screening of lung preservation solutions and their components using quantifiable biochemical endpoints. Using this model, we have developed a novel solution that improves the biochemical preservation of lung slices during cold storage. Copyright 2000 Academic Press.

[Primary sclerosing cholangitis associated with Sjögren's syndrome, retroperitoneal fibrosis and chronic pancreatitis. Report of a case].

PubMed

Barreda, F; Contardo, C; León, A; Navarrete, J; Figueroa, R; Attanasio, F

1989-01-01

Primary Sclerosing Cholangitis (PSC) is an unusual chronic, cholestatic disease of unknown etiology, more frequently seen in young adults in close relationship with Chronic Ulcerative Colitis. We report the case of a 30 year old woman, coming from the peruvian amazon with PSC associated with Sjögren Syndrome, Chronic Pancreatitis and Retroperitoneal Fibrosis, without colonic involvement. She was treated with external biliary drainage and controlled for 12 months. In this paper, clinical, biochemical, radiological, histological and therapeutic features are reviewed as well as its possible immunologie autoimmune origin.

Chronic systemic pesticide exposure reproduces features of Parkinson's disease.

PubMed

Betarbet, R; Sherer, T B; MacKenzie, G; Garcia-Osuna, M; Panov, A V; Greenamyre, J T

2000-12-01

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Randomizing Genome-Scale Metabolic Networks

PubMed Central

Samal, Areejit; Martin, Olivier C.

2011-01-01

Networks coming from protein-protein interactions, transcriptional regulation, signaling, or metabolism may appear to have “unusual” properties. To quantify this, it is appropriate to randomize the network and test the hypothesis that the network is not statistically different from expected in a motivated ensemble. However, when dealing with metabolic networks, the randomization of the network using edge exchange generates fictitious reactions that are biochemically meaningless. Here we provide several natural ensembles of randomized metabolic networks. A first constraint is to use valid biochemical reactions. Further constraints correspond to imposing appropriate functional constraints. We explain how to perform these randomizations with the help of Markov Chain Monte Carlo (MCMC) and show that they allow one to approach the properties of biological metabolic networks. The implication of the present work is that the observed global structural properties of real metabolic networks are likely to be the consequence of simple biochemical and functional constraints. PMID:21779409

Endosymbiotic associations within protists

PubMed Central

Nowack, Eva C. M.; Melkonian, Michael

2010-01-01

The establishment of an endosymbiotic relationship typically seems to be driven through complementation of the host's limited metabolic capabilities by the biochemical versatility of the endosymbiont. The most significant examples of endosymbiosis are represented by the endosymbiotic acquisition of plastids and mitochondria, introducing photosynthesis and respiration to eukaryotes. However, there are numerous other endosymbioses that evolved more recently and repeatedly across the tree of life. Recent advances in genome sequencing technology have led to a better understanding of the physiological basis of many endosymbiotic associations. This review focuses on endosymbionts in protists (unicellular eukaryotes). Selected examples illustrate the incorporation of various new biochemical functions, such as photosynthesis, nitrogen fixation and recycling, and methanogenesis, into protist hosts by prokaryotic endosymbionts. Furthermore, photosynthetic eukaryotic endosymbionts display a great diversity of modes of integration into different protist hosts. In conclusion, endosymbiosis seems to represent a general evolutionary strategy of protists to acquire novel biochemical functions and is thus an important source of genetic innovation. PMID:20124339

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

4. VIEW NORTHEAST, radar tower (unknown function), prime search radar ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

4. VIEW NORTHEAST, radar tower (unknown function), prime search radar tower, emergency power building, and height finder radar tower - Fort Custer Military Reservation, P-67 Radar Station, .25 mile north of Dickman Road, east of Clark Road, Battle Creek, Calhoun County, MI

Computed Energetics of Nucleotides in Spatial Ribozyme Structures: An Accurate Identification of Functional Regions from Structure

PubMed Central

Torshin, Ivan Y.

2004-01-01

Ribozymes are functionally diverse RNA molecules with intrinsic catalytic activity. Multiple structural and biochemical studies are required to establish which nucleotide bases are involved in the catalysis. The relative energetic properties of the nucleotide bases have been analyzed in a set of the known ribozyme structures. It was found that many of the known catalytic nucleotides can be identified using only the structure without any additional biochemical data. The results of the calculations compare well with the available biochemical data on RNA stability. Extensive in silico mutagenesis suggests that most of the nucleotides in ribozymes stabilize the RNA. The calculations show that relative contribution of the catalytic bases to RNA stability observably differs from contributions of the noncatalytic bases. Distinction between the concepts of “relative stability” and “mutational stability” is suggested. As results of prediction for several models of ribozymes appear to be in agreement with the published data on the potential active site regions, the method can potentially be used for prediction of functional nucleotides from nucleic sequence. PMID:15105962

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

PubMed

Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis

PubMed Central

Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities. PMID:22639603

Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity.

PubMed

Van Mooy, Benjamin A S; Fredricks, Helen F; Pedler, Byron E; Dyhrman, Sonya T; Karl, David M; Koblízek, Michal; Lomas, Michael W; Mincer, Tracy J; Moore, Lisa R; Moutin, Thierry; Rappé, Michael S; Webb, Eric A

2009-03-05

Phosphorus is an obligate requirement for the growth of all organisms; major biochemical reservoirs of phosphorus in marine plankton include nucleic acids and phospholipids. However, eukaryotic phytoplankton and cyanobacteria (that is, 'phytoplankton' collectively) have the ability to decrease their cellular phosphorus content when phosphorus in their environment is scarce. The biochemical mechanisms that allow phytoplankton to limit their phosphorus demand and still maintain growth are largely unknown. Here we show that phytoplankton, in regions of oligotrophic ocean where phosphate is scarce, reduce their cellular phosphorus requirements by substituting non-phosphorus membrane lipids for phospholipids. In the Sargasso Sea, where phosphate concentrations were less than 10 nmol l-1, we found that only 1.3 +/- 0.6% of phosphate uptake was used for phospholipid synthesis; in contrast, in the South Pacific subtropical gyre, where phosphate was greater than 100 nmol l-1, plankton used 17 6% (ref. 6). Examination of the planktonic membrane lipids at these two locations showed that classes of sulphur- and nitrogen-containing membrane lipids, which are devoid of phosphorus, were more abundant in the Sargasso Sea than in the South Pacific. Furthermore, these non-phosphorus, 'substitute lipids' were dominant in phosphorus-limited cultures of all of the phytoplankton species we examined. In contrast, the marine heterotrophic bacteria we examined contained no substitute lipids and only phospholipids. Thus heterotrophic bacteria, which compete with phytoplankton for nutrients in oligotrophic regions like the Sargasso Sea, appear to have a biochemical phosphorus requirement that phytoplankton avoid by using substitute lipids. Our results suggest that phospholipid substitutions are fundamental biochemical mechanisms that allow phytoplankton to maintain growth in the face of phosphorus limitation.

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

PubMed

Huber, R; Burggraf, S; Mayer, T; Barns, S M; Rossnagel, P; Stetter, K O

1995-07-06

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Making a nutritional assessment.

PubMed Central

Pencharz, P. B.

1982-01-01

The assessment of nutritional deficiencies depends on both clinical and laboratory diagnosis. The standard physical examination should be supplemented by nutritional anthropometry, consisting of accurate growth and skinfold measurements. A careful dietary history, preferably taken by a dietitian, is necessary to construct a record of past nutrient intake. Since biochemical abnormalities often appear before clinical signs of nutritional deficiency a battery of biochemical tests is sometimes needed. In unusual cases newer techniques of assessing body composition or immunologic or physiologic function may be required. In all cases the patient's physical state, nutritional intake and biochemical status must be related to age and sex standards. PMID:7139499

Multivariate analysis of fatty acid and biochemical constitutes of seaweeds to characterize their potential as bioresource for biofuel and fine chemicals.

PubMed

Verma, Priyanka; Kumar, Manoj; Mishra, Girish; Sahoo, Dinabandhu

2017-02-01

In the present study bio prospecting of thirty seaweeds from Indian coasts was analyzed for their biochemical components including pigments, fatty acid and ash content. Multivariate analysis of biochemical components and fatty acids was done using Principal Component Analysis (PCA) and Agglomerative hierarchical clustering (AHC) to manifest chemotaxonomic relationship among various seaweeds. The overall analysis suggests that these seaweeds have multi-functional properties and can be utilized as promising bioresource for proteins, lipids, pigments and carbohydrates for the food/feed and biofuel industry. Copyright © 2016. Published by Elsevier Ltd.

Metabonomics and medicine: the Biochemical Oracle.

PubMed

Mitchell, Steve; Holmes, Elaine; Carmichael, Paul

2002-10-01

Occasionally, a new idea emerges that has the potential to revolutionize an entire field of scientific endeavour. It is now within our grasp to be able to detect subtle perturbations within the phenomenally complex biochemical matrix of living organisms. The discipline of metabonomics promises an all-encompassing approach to understanding total, yet fundamental, changes occurring in disease processes, drug toxicity and cell function.

Physiologic and biochemical aspects of skeletal muscle denervation and reinnervation

NASA Technical Reports Server (NTRS)

Max, S. R.; Mayer, R. F.

1984-01-01

Some of the physiologic and biochemical changes that occur in mammalian skeletal muscle following denervation and reinnervation are considered and some comparisons are made with changes observed following altered motor function. The nature of the trophic influence by which nerves control muscle properties are discussed, including the effects of choline acetyltransferase and acetylcholinesterase and the role of the acetylcholine receptor.

Evolutionary, structural and biochemical evidence for a new interaction site of the leptin obesity protein

NASA Technical Reports Server (NTRS)

Gaucher, Eric A.; Miyamoto, Michael M.; Benner, Steven A.

2003-01-01

The Leptin protein is central to the regulation of energy metabolism in mammals. By integrating evolutionary, structural, and biochemical information, a surface segment, outside of its known receptor contacts, is predicted as a second interaction site that may help to further define its roles in energy balance and its functional differences between humans and other mammals.

Stochastic hybrid systems for studying biochemical processes.

PubMed

Singh, Abhyudai; Hespanha, João P

2010-11-13

Many protein and mRNA species occur at low molecular counts within cells, and hence are subject to large stochastic fluctuations in copy numbers over time. Development of computationally tractable frameworks for modelling stochastic fluctuations in population counts is essential to understand how noise at the cellular level affects biological function and phenotype. We show that stochastic hybrid systems (SHSs) provide a convenient framework for modelling the time evolution of population counts of different chemical species involved in a set of biochemical reactions. We illustrate recently developed techniques that allow fast computations of the statistical moments of the population count, without having to run computationally expensive Monte Carlo simulations of the biochemical reactions. Finally, we review different examples from the literature that illustrate the benefits of using SHSs for modelling biochemical processes.

A master equation and moment approach for biochemical systems with creation-time-dependent bimolecular rate functions

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

Chevalier, Michael W., E-mail: Michael.Chevalier@ucsf.edu; El-Samad, Hana, E-mail: Hana.El-Samad@ucsf.edu

Noise and stochasticity are fundamental to biology and derive from the very nature of biochemical reactions where thermal motion of molecules translates into randomness in the sequence and timing of reactions. This randomness leads to cell-to-cell variability even in clonal populations. Stochastic biochemical networks have been traditionally modeled as continuous-time discrete-state Markov processes whose probability density functions evolve according to a chemical master equation (CME). In diffusion reaction systems on membranes, the Markov formalism, which assumes constant reaction propensities is not directly appropriate. This is because the instantaneous propensity for a diffusion reaction to occur depends on the creation timesmore » of the molecules involved. In this work, we develop a chemical master equation for systems of this type. While this new CME is computationally intractable, we make rational dimensional reductions to form an approximate equation, whose moments are also derived and are shown to yield efficient, accurate results. This new framework forms a more general approach than the Markov CME and expands upon the realm of possible stochastic biochemical systems that can be efficiently modeled.« less

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors

PubMed Central

Takashima, Eizo; Williams, Marni; Eiglmeier, Karin; Pain, Adrien; Guelbeogo, Wamdaogo M.; Gneme, Awa; Brito-Fravallo, Emma; Holm, Inge; Lavazec, Catherine; Sagnon, N’Fale; Baxter, Richard H.; Riehle, Michelle M.; Vernick, Kenneth D.

2015-01-01

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity. PMID:26633695

Using kisspeptin to assess GnRH function in an unusual case of primary amenorrhoea.

PubMed

Vimalesvaran, S; Narayanaswamy, S; Yang, L; Prague, J K; Buckley, A; Miras, A D; Franks, S; Meeran, K; Dhillo, W S

2017-01-01

Primary amenorrhoea is defined as the failure to commence menstruation by the age of 15 years, in the presence of normal secondary sexual development. The potential causes of primary amenorrhoea extend from structural to chromosomal abnormalities. Polycystic ovarian syndrome (PCOS) is a common cause of secondary amenorrhoea but an uncommon cause of primary amenorrhoea. An early and prompt diagnosis of PCOS is important, as up to 30% of these women are predisposed to glucose intolerance and obesity, with the subgroup of women presenting with primary amenorrhoea and PCOS displaying a higher incidence of metabolic dysfunction. We describe a case of an 18-year-old female presenting with primary amenorrhoea of unknown aetiology. Although initial investigations did not demonstrate clinical or biochemical hyperandrogenism or any radiological evidence of polycystic ovaries, a raised luteinising hormone (LH) suggested a diagnosis of PCOS. If PCOS was the correct diagnosis, then one would expect intact hypothalamic GnRH and pituitary gonadotropin release. We used the novel hormone kisspeptin to confirm intact hypothalamic GnRH release and a GnRH stimulation test to confirm intact pituitary gonadotroph function. This case highlights that kisspeptin is a potential unique tool to test GnRH function in patients presenting with reproductive disorders. Polycystic ovarian syndrome (PCOS) can present with primary amenorrhoea, and therefore, should be considered in the differential diagnosis.PCOS is a heterogeneous condition that may present in lean women with few or absent signs of hyperandrogenism.GnRH stimulation tests are useful in evaluating pituitary function; however, to date, we do not have a viable test of GnRH function. Kisspeptin has the potential to form a novel diagnostic tool for assessing hypothalamic GnRH function by monitoring gonadotropin response as a surrogate marker of GnRH release.Confirmation of intact GnRH function helps consolidate a diagnosis in primary amenorrhoea and gives an indication of future fertility.

GDSL lipases modulate immunity through lipid homeostasis in rice

PubMed Central

Lam, Sin Man; Tong, Xiaohong; Liu, Jiyun; Wang, Xin; Shui, Guanghou

2017-01-01

Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity. PMID:29131851

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

PubMed

Tikka, Saara; Monogioudi, Evanthia; Gotsopoulos, Athanasios; Soliymani, Rabah; Pezzini, Francesco; Scifo, Enzo; Uusi-Rauva, Kristiina; Tyynelä, Jaana; Baumann, Marc; Jalanko, Anu; Simonati, Alessandro; Lalowski, Maciej

2016-03-01

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood encephalopathy.

Evolution and functional characterisation of melanopsins in a deep-sea chimaera (elephant shark, Callorhinchus milii).

PubMed

Davies, Wayne I L; Tay, Boon-Hui; Zheng, Lei; Danks, Janine A; Brenner, Sydney; Foster, Russell G; Collin, Shaun P; Hankins, Mark W; Venkatesh, Byrappa; Hunt, David M

2012-01-01

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas "long" and "short" splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both "invertebrate-like" bistable and classical "vertebrate-like" monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.

Evolution and Functional Characterisation of Melanopsins in a Deep-Sea Chimaera (Elephant Shark, Callorhinchus milii)

PubMed Central

Davies, Wayne I. L.; Tay, Boon-Hui; Zheng, Lei; Danks, Janine A.; Brenner, Sydney; Foster, Russell G.; Collin, Shaun P.; Hankins, Mark W.; Venkatesh, Byrappa; Hunt, David M.

2012-01-01

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas “long” and “short” splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both “invertebrate-like” bistable and classical “vertebrate-like” monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates. PMID:23251480

Murine but not human basophil undergoes cell-specific proteolysis of a major endoplasmic reticulum chaperone.

PubMed

Liu, Bei; Staron, Matthew; Li, Zihai

2012-01-01

Basophil has been implicated in anti-parasite defense, allergy and in polarizing T(H)2 response. Mouse model has been commonly used to study basophil function although the difference between human and mouse basophils is underappreciated. As an essential chaperone for multiple Toll-like receptors and integrins in the endoplasmic reticulum, gp96 also participates in general protein homeostasis and in the ER unfolded protein response to ensure cell survival during stress. The roles of gp96 in basophil development are unknown. We genetically delete gp96 in mice and examined the expression of gp96 in basophils by Western blot and flow cytometry. We compared the expression pattern of gp96 between human and mouse basophils. We found that gp96 was dispensable for murine basophil development. Moreover, gp96 was cleaved by serine protease(s) in murine but not human basophils leading to accumulation of a nun-functional N-terminal ∼50 kDa fragment and striking induction of the unfolded protein response. The alteration of gp96 was unique to basophils and was not observed in any other cell types including mast cells. We also demonstrated that the ectopic expression of a mouse-specific tryptase mMCP11 does not lead to gp96 cleavage in human basophils. Our study revealed a remarkable biochemical event of gp96 silencing in murine but not human basophils, highlighting the need for caution in using mouse models to infer the function of basophils in human immune response. Our study also reveals a novel mechanism of shutting down gp96 post-translationally in regulating its function.

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

PubMed

Kisly, Ivan; Gulay, Suna P; Mäeorg, Uno; Dinman, Jonathan D; Remme, Jaanus; Tamm, Tiina

2016-05-22

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Long-term biological investigations in space.

PubMed

Lotz, R G; Fuchs, H; Bertsche, U

1975-01-01

Missions in space within the next two decades will be of longer duration than those carried out up to the present time, and the effects of such long-term flights on biological organisms are unknown. Results of biological experiments that have been performed to date cannot be extrapolated to results in future flights because of the unknown influence of adaptation over a long period of time. Prior experiments with Axolotl, fishes, and vertebrates by our research team (in part with sounding rockets) showed that these specimens did not appear to be suitable for long-term missions on which minimization of expense, technique, and energy is required. Subsequent investigations have shown the suitability of the leech (Hirudo medicinalis), which consumes blood of mammals up to ten times its own weight (1 g) and can live more than 2 years without further food supply. Emphasis in the experiments with Hirudo medicinalis is placed on metabolic rhythm and motility. Resorption and diffusion in tissue, development, and growth under long-term effects of cosmic proton radiation and zero-gravity are other focal points. The constancy of cellular life in the mature animals is a point in favor of these specimens. We have also taken into account the synergistic effects of the space environment on the problems just mentioned. The life-support system constructed for the leech has been tested successfully in four sounding rocket flights and, on that basis, has been prepared for a long-term mission. Long-term investigations out of the terrestrial biosphere will provide us with information concerning the degree of adaptation of certain physiological and biochemical functions and as to what extent biological readjustment or repair processes can occur under the specific stress conditions of space flight.

Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment.

PubMed

Rabus, Ralf; Boll, Matthias; Heider, Johann; Meckenstock, Rainer U; Buckel, Wolfgang; Einsle, Oliver; Ermler, Ulrich; Golding, Bernard T; Gunsalus, Robert P; Kroneck, Peter M H; Krüger, Martin; Lueders, Tillmann; Martins, Berta M; Musat, Florin; Richnow, Hans H; Schink, Bernhard; Seifert, Jana; Szaleniec, Maciej; Treude, Tina; Ullmann, G Matthias; Vogt, Carsten; von Bergen, Martin; Wilkes, Heinz

2016-01-01

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl)succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons. © 2016 S. Karger AG, Basel.

The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.

PubMed

Jacobsen, Annette V; Murphy, James M

2017-06-15

Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic diversity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

PubMed Central

Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2010-01-01

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

FUSION-Guided Hypothesis Development Leads to the Identification of N6,N6-Dimethyladenosine, a Marine-Derived AKT Pathway Inhibitor

PubMed Central

Vaden, Rachel M.; Oswald, Nathaniel W.; Potts, Malia B.; MacMillan, John B.; White, Michael A.

2017-01-01

Chemicals found in nature have evolved over geological time scales to productively interact with biological molecules, and thus represent an effective resource for pharmaceutical development. Marine-derived bacteria are rich sources of chemically diverse, bioactive secondary metabolites, but harnessing this diversity for biomedical benefit is limited by challenges associated with natural product purification and determination of biochemical mechanism. Using Functional Signature Ontology (FUSION), we report the parallel isolation and characterization of a marine-derived natural product, N6,N6-dimethyladenosine, that robustly inhibits AKT signaling in a variety of non-small cell lung cancer cell lines. Upon validation of the elucidated structure by comparison with a commercially available sample, experiments were initiated to understand the small molecule’s breadth of effect in a biological setting. One such experiment, a reverse phase protein array (RPPA) analysis of >50 kinases, indicated a specific cellular response to treatment. In all, leveraging the FUSION platform allowed for the rapid generation and validation of a biological mechanism of action hypothesis for an unknown natural product and permitted accelerated purification of the bioactive component from a chemically complex fraction. PMID:28294973

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

PubMed Central

Zhou, Yihua; Xu, Bixiong C.; Maheshwari, Hiralal G.; He, Li; Reed, Michael; Lozykowski, Maria; Okada, Shigeru; Cataldo, Lori; Coschigamo, Karen; Wagner, Thomas E.; Baumann, Gerhard; Kopchick, John J.

1997-01-01

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans. PMID:9371826

Intraneuronal Amyloid Beta Accumulation Disrupts Hippocampal CRTC1-Dependent Gene Expression and Cognitive Function in a Rat Model of Alzheimer Disease

PubMed Central

Wilson, Edward N.; Abela, Andrew R.; Do Carmo, Sonia; Allard, Simon; Marks, Adam R.; Welikovitch, Lindsay A.; Ducatenzeiler, Adriana; Chudasama, Yogita; Cuello, A. Claudio

2017-01-01

In Alzheimer disease (AD), the accumulation of amyloid beta (Aβ) begins decades before cognitive symptoms and progresses from intraneuronal material to extracellular plaques. To date, however, the precise mechanism by which the early buildup of Aβ peptides leads to cognitive dysfunction remains unknown. Here, we investigate the impact of the early Aβ accumulation on temporal and frontal lobe dysfunction. We compared the performance of McGill-R-Thy1-APP transgenic AD rats with wild-type littermate controls on a visual discrimination task using a touchscreen operant platform. Subsequently, we conducted studies to establish the biochemical and molecular basis for the behavioral alterations. It was found that the presence of intraneuronal Aβ caused a severe associative learning deficit in the AD rats. This coincided with reduced nuclear translocation and genomic occupancy of the CREB co-activator, CRTC1, and decreased production of synaptic plasticity-associated transcripts Arc, c-fos, Egr1, and Bdnf. Thus, blockade of CRTC1-dependent gene expression in the early, preplaque phase of AD-like pathology provides a molecular basis for the cognitive deficits that figure so prominently in early AD. PMID:26759481

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

PubMed

Zhou, Y; Xu, B C; Maheshwari, H G; He, L; Reed, M; Lozykowski, M; Okada, S; Cataldo, L; Coschigamo, K; Wagner, T E; Baumann, G; Kopchick, J J

1997-11-25

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Digitalis metabolism and human liver alcohol dehydrogenase.

PubMed Central

Frey, W A; Vallee, B L

1980-01-01

Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

Energy parasites trigger oncogene mutation.

PubMed

Pokorný, Jiří; Pokorný, Jan; Jandová, Anna; Kobilková, Jitka; Vrba, Jan; Vrba, Jan

2016-10-01

Cancer initialization can be explained as a result of parasitic virus energy consumption leading to randomized genome chemical bonding. Analysis of experimental data on cell-mediated immunity (CMI) containing about 12,000 cases of healthy humans, cancer patients and patients with precancerous cervical lesions disclosed that the specific cancer and the non-specific lactate dehydrogenase-elevating (LDH) virus antigen elicit similar responses. The specific antigen is effective only in cancer type of its origin but the non-specific antigen in all examined cancers. CMI results of CIN patients display both healthy and cancer state. The ribonucleic acid (RNA) of the LDH virus parasitizing on energy reduces the ratio of coherent/random oscillations. Decreased effect of coherent cellular electromagnetic field on bonding electrons in biological macromolecules leads to elevating probability of random genome reactions. Overlapping of wave functions in biological macromolecules depends on energy of the cellular electromagnetic field which supplies energy to bonding electrons for selective chemical bonds. CMI responses of cancer and LDH virus antigens in all examined healthy, precancerous and cancer cases point to energy mechanism in cancer initiation. Dependence of the rate of biochemical reactions on biological electromagnetic field explains yet unknown mechanism of genome mutation.

Mercury and autism: accelerating evidence?

PubMed

Mutter, Joachim; Naumann, Johannes; Schneider, Rainer; Walach, Harald; Haley, Boyd

2005-10-01

The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury exposure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a correlation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymorphism. In vitro, mercury and thimerosal in levels found several days after vaccination inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain development, attention and production of glutathione, an important antioxidative and detoxifying agent. Repetitive doses of thimerosal leads to neurobehavioral deteriorations in autoimmune susceptible mice, increased oxidative stress and decreased intracellular levels of glutathione in vitro. Subsequently, autistic children have significantly decreased level of reduced glutathione. Promising treatments of autism involve detoxification of mercury, and supplementation of deficient metabolites.

Protein arginine deiminase 2 binds calcium in an ordered fashion: Implications for inhibitor design

DOE PAGES

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; ...

2015-01-26

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design

PubMed Central

2015-01-01

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs. PMID:25621824

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines1[OPEN

PubMed Central

Martinis, Jacopo; Gas-Pascual, Elisabet; Szydlowski, Nicolas; Crèvecoeur, Michèle; Gisler, Alexandra; Bürkle, Lukas; Fitzpatrick, Teresa B.

2016-01-01

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings. PMID:27006489

Emerging concepts promising new horizons for marine biodiscovery and synthetic biology.

PubMed

Reen, F Jerry; Gutiérrez-Barranquero, José A; Dobson, Alan D W; Adams, Claire; O'Gara, Fergal

2015-05-13

The vast oceans of the world, which comprise a huge variety of unique ecosystems, are emerging as a rich and relatively untapped source of novel bioactive compounds with invaluable biotechnological and pharmaceutical potential. Evidence accumulated over the last decade has revealed that the diversity of marine microorganisms is enormous with many thousands of bacterial species detected that were previously unknown. Associated with this diversity is the production of diverse repertoires of bioactive compounds ranging from peptides and enzymes to more complex secondary metabolites that have significant bioactivity and thus the potential to be exploited for innovative biotechnology. Here we review the discovery and functional potential of marine bioactive peptides such as lantibiotics, nanoantibiotics and peptidomimetics, which have received particular attention in recent years in light of their broad spectrum of bioactivity. The significance of marine peptides in cell-to-cell communication and how this may be exploited in the discovery of novel bioactivity is also explored. Finally, with the recent advances in bioinformatics and synthetic biology, it is becoming clear that the integration of these disciplines with genetic and biochemical characterization of the novel marine peptides, offers the most potential in the development of the next generation of societal solutions.

Emerging Concepts Promising New Horizons for Marine Biodiscovery and Synthetic Biology

PubMed Central

Reen, F. Jerry; Gutiérrez-Barranquero, José A.; Dobson, Alan D. W.; Adams, Claire; O’Gara, Fergal

2015-01-01

The vast oceans of the world, which comprise a huge variety of unique ecosystems, are emerging as a rich and relatively untapped source of novel bioactive compounds with invaluable biotechnological and pharmaceutical potential. Evidence accumulated over the last decade has revealed that the diversity of marine microorganisms is enormous with many thousands of bacterial species detected that were previously unknown. Associated with this diversity is the production of diverse repertoires of bioactive compounds ranging from peptides and enzymes to more complex secondary metabolites that have significant bioactivity and thus the potential to be exploited for innovative biotechnology. Here we review the discovery and functional potential of marine bioactive peptides such as lantibiotics, nanoantibiotics and peptidomimetics, which have received particular attention in recent years in light of their broad spectrum of bioactivity. The significance of marine peptides in cell-to-cell communication and how this may be exploited in the discovery of novel bioactivity is also explored. Finally, with the recent advances in bioinformatics and synthetic biology, it is becoming clear that the integration of these disciplines with genetic and biochemical characterization of the novel marine peptides, offers the most potential in the development of the next generation of societal solutions. PMID:25984990

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

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

Kim, Jeong Sig; Ro, Seung-Hyun; Kim, Myungjin

2015-03-30

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1more » subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.« less

PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1.

PubMed

Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki; Roeder, Robert G

2015-02-01

PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. © 2015 Iida et al.; Published by Cold Spring Harbor Laboratory Press.

Nonspecific uptake and homeostasis drive the oceanic cadmium cycle

NASA Astrophysics Data System (ADS)

Horner, Tristan J.; Lee, Renee B. Y.; Henderson, Gideon M.; Rickaby, Rosalind E. M.

2013-02-01

The global marine distributions of Cd and phosphate are closely correlated, which has led to Cd being considered as a marine micronutrient, despite its toxicity to life. The explanation for this nutrient-like behavior is unknown because there is only one identified biochemical function for Cd, an unusual Cd/Zn carbonic anhydrase. Recent developments in Cd isotope mass spectrometry have revealed that Cd uptake by phytoplankton causes isotopic fractionation in the open ocean and in culture. Here we investigate the physiochemical pathways that fractionate Cd isotopes by performing subcellular Cd isotope analysis on genetically modified microorganisms. We find that expression of the Cd/Zn carbonic anhydrase makes no difference to the Cd isotope composition of whole cells. Instead, a large proportion of the Cd is partitioned into cell membranes with a similar direction and magnitude of Cd isotopic fractionation to that seen in surface seawater. This observation is well explained if Cd is mistakenly imported with other divalent metals and subsequently managed by binding within the cell to avoid toxicity. This process may apply to other divalent metals, whereby nonspecific uptake and subsequent homeostasis may contribute to elemental and isotopic distributions in seawater, even for elements commonly considered as micronutrients.

S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

PubMed

Lv, Zongyang; Rickman, Kimberly A; Yuan, Lingmin; Williams, Katelyn; Selvam, Shanmugam Panneer; Woosley, Alec N; Howe, Philip H; Ogretmen, Besim; Smogorzewska, Agata; Olsen, Shaun K

2017-02-16

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunology of scorpion toxins and perspectives for generation of anti-venom vaccines.

PubMed

Gazarian, Karlen G; Gazarian, Tatiana; Hernández, Ricardo; Possani, Lourival D

2005-05-16

Scorpions and other venomous animals contain concentrates of biologically active substances developed to block vital physiological and biochemical functions of the victims. These have contrasting human health concerns, provide important pharmacological raw material and pose a serious threat to human life and health in tropical and subtropical regions. Because only occasional and minor quantities of venom are introduced into the human organism with a scorpion sting and their mortal effect is an acute phenomenon these substances are unknown to the immune defense system and thus no immunity has appeared against them during evolution. Antidotes prepared from animal anti-sera are effective against some species of scorpions but depend on the manufacturer and the availability of product to the medical community. Although significant progress has been made in immunological studies of certain groups of toxins, few centers are dedicated to this research. Information is still insufficient to generate a comprehensive picture of the subject and to propose vaccines against venoms. A novel approach based on mimotopes selected from phage-displayed random peptide libraries show potential to impel further progress of toxin immunological studies and to provide putative vaccine resources. In this report we revise the "state of the art" in the field.

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E

2013-01-01

By coupling the protection and organization of ssDNA with the recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA manages to coordinate the biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA s DNA binding activity, combining small-angle x-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA s DNA-binding core. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than threemore » transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.« less

Effects of short-term hormonal replacement on learning and on basal forebrain ChAT and TrkA content in ovariectomized rats.

PubMed

Espinosa-Raya, Judith; Plata-Cruz, Noemí; Neri-Gómez, Teresa; Camacho-Arroyo, Ignacio; Picazo, Ofir

2011-02-23

It has been proposed that sex steroid hormones improve performance in some cognitive tasks by regulating the basal forebrain cholinergic function. However, the molecular basis of such influence still remains unknown. Current study analyzed the performance of ovariectomized rats in an autoshaping learning task after a short-term treatment with 17β-estradiol (E2: 4 and 40μg/kg) and/or progesterone (P4: 4mg/kg). These results were correlated with basal forebrain choline acetyltransferase (ChAT) and TrkA protein content. The high dose of E2 enhanced both acquisition in the autoshaping task and the content of ChAT and TrkA. P4 treatment increased ChAT and TrkA content without affecting performance of rats in the autoshaping learning task. Interestingly, the continuous and simultaneous administration of E2 plus P4 did not significantly modify behavioral and biochemical evaluated parameters. These results address the influence of both E2 and P4 on cholinergic and TrkA activity and suggest that the effects of ovarian hormones on cognitive performance involve basal forebrain cholinergic neurons. Copyright © 2010 Elsevier B.V. All rights reserved.

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

How to investigate neuro-biochemical relationships on a regional level in humans? Methodological considerations for combining functional with biochemical imaging.

PubMed

Duncan, Niall W; Wiebking, Christine; Muñoz-Torres, Zeidy; Northoff, Georg

2014-01-15

There is an increasing interest in combining different imaging modalities to investigate the relationship between neural and biochemical activity. More specifically, imaging techniques like MRS and PET that allow for biochemical measurement are combined with techniques like fMRI and EEG that measure neural activity in different states. Such combination of neural and biochemical measures raises not only technical issues, such as merging the different data sets, but also several methodological issues. These methodological issues – ranging from hypothesis generation and hypothesis-guided use of technical facilities to target measures and experimental measures – are the focus of this paper. We discuss the various methodological problems and issues raised by the combination of different imaging methodologies in order to investigate neuro-biochemical relationships on a regional level in humans. For example, the choice of transmitter and scan type is discussed, along with approaches to allow the establishment of particular specificities (such as regional or biochemical) to in turn make results fully interpretable. An algorithm that can be used as a form of checklist for designing such multimodal studies is presented. The paper concludes that while several methodological and technical caveats needs to be overcome and addressed, multimodal imaging of the neuro-biochemical relationship provides an important tool to better understand the physiological mechanisms of the human brain.

How to investigate neuro-biochemical relationships on a regional level in humans? Methodological considerations for combining functional with biochemical imaging.

PubMed

Duncan, Niall W; Wiebking, Christine; Munoz-Torres, Zeidy; Northoff, Georg

2013-10-25

There is an increasing interest in combining different imaging modalities to investigate the relationship between neural and biochemical activity. More specifically, imaging techniques like MRS and PET that allow for biochemical measurement are combined with techniques like fMRI and EEG that measure neural activity in different states. Such combination of neural and biochemical measures raises not only technical issues, such as merging the different data sets, but also several methodological issues. These methodological issues - ranging from hypothesis generation and hypothesis-guided use of technical facilities to target measures and experimental measures - are the focus of this paper. We discuss the various methodological problems and issues raised by the combination of different imaging methodologies in order to investigate neuro-biochemical relationships on a regional level in humans. For example, the choice of transmitter and scan type is discussed, along with approaches to allow the establishment of particular specificities (such as regional or biochemical) to in turn make results fully interpretable. An algorithm that can be used as a form of checklist for designing such multimodal studies is presented. The paper concludes that while several methodological and technical caveats needs to be overcome and addressed, multimodal imaging of the neuro-biochemical relationship provides an important tool to better understand the physiological mechanisms of the human brain. Copyright © 2013. Published by Elsevier B.V.

Adaptive Fault-Tolerant Control of Uncertain Nonlinear Large-Scale Systems With Unknown Dead Zone.

PubMed

Chen, Mou; Tao, Gang

2016-08-01

In this paper, an adaptive neural fault-tolerant control scheme is proposed and analyzed for a class of uncertain nonlinear large-scale systems with unknown dead zone and external disturbances. To tackle the unknown nonlinear interaction functions in the large-scale system, the radial basis function neural network (RBFNN) is employed to approximate them. To further handle the unknown approximation errors and the effects of the unknown dead zone and external disturbances, integrated as the compounded disturbances, the corresponding disturbance observers are developed for their estimations. Based on the outputs of the RBFNN and the disturbance observer, the adaptive neural fault-tolerant control scheme is designed for uncertain nonlinear large-scale systems by using a decentralized backstepping technique. The closed-loop stability of the adaptive control system is rigorously proved via Lyapunov analysis and the satisfactory tracking performance is achieved under the integrated effects of unknown dead zone, actuator fault, and unknown external disturbances. Simulation results of a mass-spring-damper system are given to illustrate the effectiveness of the proposed adaptive neural fault-tolerant control scheme for uncertain nonlinear large-scale systems.

Increased dysbindin-1B isoform expression in schizophrenia and its propensity in aggresome formation

PubMed Central

Xu, Yiliang; Sun, Yuhui; Ye, Haihong; Zhu, Li; Liu, Jianghong; Wu, Xiaofeng; Wang, Le; He, Tingting; Shen, Yan; Wu, Jane Y; Xu, Qi

2015-01-01

Genetic variations in the human dysbindin-1 gene (DTNBP1) have been associated with schizophrenia. As a result of alternative splicing, the human DTNBP1 gene generates at least three distinct protein isoforms, dysbindin-1A, -1B and -1C. Significant effort has focused on dysbindin-1A, an important player in multiple steps of neurodevelopment. However, the other isoforms, dysbindin-1B and dysbindin-1C have not been well characterized. Nor have been associated with human diseases. Here we report an increase in expression of DTNBP1b mRNA in patients with paranoid schizophrenia as compared with healthy controls. A single-nucleotide polymorphism located in intron 9, rs117610176, has been identified and associated with paranoid schizophrenia, and its C allele leads to an increase of DTNBP1b mRNA splicing. Our data show that different dysbindin splicing isoforms exhibit distinct subcellular distribution, suggesting their distinct functional activities. Dysbindin-1B forms aggresomes at the perinuclear region, whereas dysbindin-1A and -1C proteins exhibit diffused patterns in the cytoplasm. Dysbindin-1A interacts with dysbindin-1B, getting recruited to the aggresome structure when co-expressed with dysbindin-1B. Moreover, cortical neurons over-expressing dysbindin-1B show reduction in neurite outgrowth, suggesting that dysbindin-1B may interfere with dysbindin-1A function in a dominant-negative manner. Taken together, our study uncovers a previously unknown association of DTNBP1b expression with schizophrenia in addition to its distinct biochemical and functional properties. PMID:27462430

Fibrin Formation, Structure and Properties

PubMed Central

Weisel, John W.; Litvinov, Rustem I.

2017-01-01

Fibrinogen and fibrin are essential for hemostasis and are major factors in thrombosis, wound healing, and several other biological functions and pathological conditions. The X-ray crystallographic structure of major parts of fibrin(ogen), together with computational reconstructions of missing portions and numerous biochemical and biophysical studies, have provided a wealth of data to interpret molecular mechanisms of fibrin formation, its organization, and properties. On cleavage of fibrinopeptides by thrombin, fibrinogen is converted to fibrin monomers, which interact via knobs exposed by fibrinopeptide removal in the central region, with holes always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers, which then branch to yield a three-dimensional network. Much is now known about the structural origins of clot mechanical properties, including changes in fiber orientation, stretching and buckling, and forced unfolding of molecular domains. Studies of congenital fibrinogen variants and post-translational modifications have increased our understanding of the structure and functions of fibrin(ogen). The fibrinolytic system, with the zymogen plasminogen binding to fibrin together with tissue-type plasminogen activator to promote activation to the active proteolytic enzyme, plasmin, results in digestion of fibrin at specific lysine residues. In spite of a great increase in our knowledge of all these interconnected processes, much about the molecular mechanisms of the biological functions of fibrin(ogen) remains unknown, including some basic aspects of clotting, fibrinolysis, and molecular origins of fibrin mechanical properties. Even less is known concerning more complex (patho)physiological implications of fibrinogen and fibrin. PMID:28101869

Human vs. Mouse Eosinophils: “That which we call an eosinophil, by any other name would stain as red”

PubMed Central

Lee, James J.; Jacobsen, Elizabeth A.; Ochkur, Sergei I; McGarry, Michael P.; Condjella, Rachel M.; Doyle, Alfred D.; Luo, Huijun; Zellner, Katie R.; Protheroe, Cheryl A.; Willetts, Lian; LeSuer, William E.; Colbert, Dana C.; Helmers, Richard A.; Lacy, Paige; Moqbel, Redwan; Lee, Nancy A.

2012-01-01

The respective life histories of humans and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiological pathways. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils and/or their effector functions. Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. The goal of this review is to summarize this literature and in some cases provide the experimental details, comparing and contrasting eosinophils and eosinophil effector functions in humans vs. mice. In particular, our review will provide a summation and an easy to use reference guide to important studies demonstrating that while differences exist, more often than not their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function, but instead, species-specific variations. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients. PMID:22935586

Structure and Dynamics of the Liver Receptor Homolog 1-PGC1α Complex.

PubMed

Mays, Suzanne G; Okafor, C Denise; Tuntland, Micheal L; Whitby, Richard J; Dharmarajan, Venkatasubramanian; Stec, Józef; Griffin, Patrick R; Ortlund, Eric A

2017-07-01

Peroxisome proliferator-activated gamma coactivator 1- α (PGC1 α ) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1 α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1 α is known to bind and activate LRH-1, mechanisms through which PGC1 α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1-PGC1 α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1 α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1-PGC1 α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1 α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1 α but promoted allosteric signaling from the helix 6/ β -sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1-PGC1 α interaction and may illuminate strategies for selective therapeutic targeting of PGC1 α -dependent LRH-1 signaling pathways. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Connecting Source with Sink: The Role of Arabidopsis AAP8 in Phloem Loading of Amino Acids1[OPEN

PubMed Central

Santiago, James P.; Tegeder, Mechthild

2016-01-01

Allocation of large amounts of nitrogen to developing organs occurs in the phloem and is essential for plant growth and seed development. In Arabidopsis (Arabidopsis thaliana) and many other plant species, amino acids represent the dominant nitrogen transport forms in the phloem, and they are mainly synthesized in photosynthetically active source leaves. Following their synthesis, a broad spectrum of the amino nitrogen is actively loaded into the phloem of leaf minor veins and transported within the phloem sap to sinks such as developing leaves, fruits, or seeds. Controlled regulation of the source-to-sink transport of amino acids has long been postulated; however, the molecular mechanism of amino acid phloem loading was still unknown. In this study, Arabidopsis AMINO ACID PERMEASE8 (AAP8) was shown to be expressed in the source leaf phloem and localized to the plasma membrane, suggesting its function in phloem loading. This was further supported by transport studies with aap8 mutants fed with radiolabeled amino acids and by leaf exudate analyses. In addition, biochemical and molecular analyses revealed alterations in leaf nitrogen pools and metabolism dependent on the developmental stage of the mutants. Decreased amino acid phloem loading and partitioning to sinks led to decreased silique and seed numbers, but seed protein levels were unchanged, demonstrating the importance of AAP8 function for sink development rather than seed quality. Overall, these results show that AAP8 plays an important role in source-to-sink partitioning of nitrogen and that its function affects source leaf physiology and seed yield. PMID:27016446

Anterior gradient 2 is induced in cutaneous wound and promotes wound healing through its adhesion domain.

PubMed

Zhu, Qi; Mangukiya, Hitesh Bhagavanbhai; Mashausi, Dhahiri Saidi; Guo, Hao; Negi, Hema; Merugu, Siva Bharath; Wu, Zhenghua; Li, Dawei

2017-09-01

Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds. © 2017 Federation of European Biochemical Societies.

Biochemical Testing of the Thyroid: TSH is the Best and, Oftentimes, Only Test Needed - A Review for Primary Care.

PubMed

Sheehan, Michael T

2016-06-01

Disorders of thyroid function are common, and screening, diagnosis, and management are often performed by primary care providers. While management of significant biochemical abnormalities is reasonably straight forward, laboratory tests only slightly outside, or even within, the normal range are becoming more difficult to appropriately manage. A large part of this increasing difficulty in appropriate management is caused by patients requesting, and even demanding, certain tests or treatments that may not be indicated. Symptoms of thyroid dysfunction are non-specific and extremely prevalent in the general population. This, along with a growing body of information available to patients via the lay press and internet suggesting that traditional thyroid function testing is not reliable, has fostered some degree of patient mistrust. Increasingly, when a physician informs a patient that their thyroid is not the cause of their symptoms, the patient is dissatisfied and even angry. This review aims to clarify the interpretation of normal and mild abnormalities of thyroid function tests by describing pituitary-thyroid physiology and through an in depth review of, arguably, the three most important biochemical tests of thyroid function: TSH, free T4, and anti-TPO antibodies. It is important for primary care providers to have an understanding of the shortcomings and proper interpretation of these tests to be better able to discuss thyroid function with their patients. © 2016 Marshfield Clinic.

Allometric constraints to inversion of canopy structure from remote sensing

NASA Astrophysics Data System (ADS)

Wolf, A.; Berry, J. A.; Asner, G. P.

2008-12-01

Canopy radiative transfer models employ a large number of vegetation architectural and leaf biochemical attributes. Studies of leaf biochemistry show a wide array of chemical and spectral diversity that suggests that several leaf biochemical constituents can be independently retrieved from multi-spectral remotely sensed imagery. In contrast, attempts to exploit multi-angle imagery to retrieve canopy structure only succeed in finding two or three of the many unknown canopy arhitectural attributes. We examine a database of over 5000 destructive tree harvests from Eurasia to show that allometry - the covariation of plant form across a broad range of plant size and canopy density - restricts the architectural diversity of plant canopies into a single composite variable ranging from young canopies with many short trees with small crowns to older canopies with fewer trees and larger crowns. Moreover, these architectural attributes are closely linked to biomass via allometric constraints such as the "self-thinning law". We use the measured variance and covariance of plant canopy architecture in these stands to drive the radiative transfer model DISORD, which employs the Li-Strahler geometric optics model. This correlations introduced in the Monte Carlo study are used to determine which attributes of canopy architecture lead to important variation that can be observed by multi-angle or multi-spectral satellite observations, using the sun-view geometry characteristic of MODIS observations in different biomes located at different latitude bands. We conclude that although multi-angle/multi-spectral remote sensing is only sensitive to some of the many unknown canopy attributes that ecologists would wish to know, the strong allometric covariation between these attributes and others permits a large number of inferrences, such as forest biomass, that will be meaningful next-generation vegetation products useful for data assimilation.

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

PubMed Central

Li, Dapeng; Baldwin, Ian T.; Gaquerel, Emmanuel

2015-01-01

Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United States—including the well-characterized Utah 30th generation inbred accession—and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis. PMID:26170304

BRENDA in 2013: integrated reactions, kinetic data, enzyme function data, improved disease classification: new options and contents in BRENDA.

PubMed

Schomburg, Ida; Chang, Antje; Placzek, Sandra; Söhngen, Carola; Rother, Michael; Lang, Maren; Munaretto, Cornelia; Ulas, Susanne; Stelzer, Michael; Grote, Andreas; Scheer, Maurice; Schomburg, Dietmar

2013-01-01

The BRENDA (BRaunschweig ENzyme DAtabase) enzyme portal (http://www.brenda-enzymes.org) is the main information system of functional biochemical and molecular enzyme data and provides access to seven interconnected databases. BRENDA contains 2.7 million manually annotated data on enzyme occurrence, function, kinetics and molecular properties. Each entry is connected to a reference and the source organism. Enzyme ligands are stored with their structures and can be accessed via their names, synonyms or via a structure search. FRENDA (Full Reference ENzyme DAta) and AMENDA (Automatic Mining of ENzyme DAta) are based on text mining methods and represent a complete survey of PubMed abstracts with information on enzymes in different organisms, tissues or organelles. The supplemental database DRENDA provides more than 910 000 new EC number-disease relations in more than 510 000 references from automatic search and a classification of enzyme-disease-related information. KENDA (Kinetic ENzyme DAta), a new amendment extracts and displays kinetic values from PubMed abstracts. The integration of the EnzymeDetector offers an automatic comparison, evaluation and prediction of enzyme function annotations for prokaryotic genomes. The biochemical reaction database BKM-react contains non-redundant enzyme-catalysed and spontaneous reactions and was developed to facilitate and accelerate the construction of biochemical models.

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

PubMed Central

Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

2012-01-01

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

Bio-recognition and functional lipidomics by glycosphingolipid transfer technology

PubMed Central

TAKI, Takao

2013-01-01

Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced. PMID:23883610

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

PubMed Central

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

2015-01-01

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level. DOI: http://dx.doi.org/10.7554/eLife.03599.001 PMID:26192964

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

Correction: Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

DOE PAGES

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; ...

2015-07-20

One central question is how specificity in cellular responses to the eukaryotic second messenger Ca 2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca 2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca 2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca 2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruplemore » mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca 2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca 2+-dependent and Ca 2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca 2+-signaling on a cellular, genetic, and biochemical level.« less

Correction: Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

DOE PAGES

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; ...

2015-07-29

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca 2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca 2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca 2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca 2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruplemore » mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca 2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca 2+-dependent and Ca 2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca 2+-signaling on a cellular, genetic, and biochemical level.« less

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

Applied spectrophotometry: analysis of a biochemical mixture.

PubMed

Trumbo, Toni A; Schultz, Emeric; Borland, Michael G; Pugh, Michael Eugene

2013-01-01

Spectrophotometric analysis is essential for determining biomolecule concentration of a solution and is employed ubiquitously in biochemistry and molecular biology. The application of the Beer-Lambert-Bouguer Lawis routinely used to determine the concentration of DNA, RNA or protein. There is however a significant difference in determining the concentration of a given species (RNA, DNA, protein) in isolation (a contrived circumstance) as opposed to determining that concentration in the presence of other species (a more realistic situation). To present the student with a more realistic laboratory experience and also to fill a hole that we believe exists in student experience prior to reaching a biochemistry course, we have devised a three week laboratory experience designed so that students learn to: connect laboratory practice with theory, apply the Beer-Lambert-Bougert Law to biochemical analyses, demonstrate the utility and limitations of example quantitative colorimetric assays, demonstrate the utility and limitations of UV analyses for biomolecules, develop strategies for analysis of a solution of unknown biomolecular composition, use digital micropipettors to make accurate and precise measurements, and apply graphing software. Copyright © 2013 Wiley Periodicals, Inc.

Streptococcus tangierensis sp. nov. and Streptococcus cameli sp. nov., two novel Streptococcus species isolated from raw camel milk in Morocco.

PubMed

Kadri, Zaina; Vandamme, Peter; Ouadghiri, Mouna; Cnockaert, Margo; Aerts, Maarten; Elfahime, El Mostafa; Farricha, Omar El; Swings, Jean; Amar, Mohamed

2015-02-01

Biochemical and molecular genetic studies were performed on two unidentified Gram-stain positive, catalase and oxidase negative, non-hemolytic Streptococcus-like organisms recovered from raw camel milk in Morocco. Phenotypic characterization and comparative 16S rRNA gene sequencing demonstrated that the two strains were highly different from each other and that they did not correspond to any recognized species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms each formed a hitherto unknown sub-line within the genus Streptococcus, displaying a close affinity with Streptococcus moroccensis, Streptococcus minor and Streptococcus ovis. DNA G+C content determination, MALDI-TOF mass spectrometry and biochemical tests demonstrated the bacterial isolates represent two novel species. Based on the phenotypic distinctiveness of the new bacteria and molecular genetic evidence, it is proposed to classify the two strains as Streptococcus tangierensis sp. nov., with CCMM B832(T) (=LMG 27683(T)) as the type strain, and Streptococcus cameli sp. nov., with CCMM B834(T) (=LMG 27685(T)) as the type strain.

The Endocrinology of Tuberculosis

PubMed Central

Obermer, Edgar

1928-01-01

It is suggested that the endocrines hold the key position in the mechanism of resistance to all infection. In man the glands of internal secretion are divided into two counterbalancing groups. It is the function of the “katabolic” group—or glands of emergency—to react to acute infections. It is the function of the “anabolic” group—or glands of conservation of energy—to counterbalance the prolonged over-action of the katabolic group in chronic infections. In chronic pulmonary tuberculosis improvement is dependent on the predominance of the anabolic group. Clinical and biochemical evidence to this effect is brought forward. The individual ductless glands are discussed with a view to summarizing the known tests for detecting their functional variations during life. It is suggested that present methods for the determination of basal metabolic rate are inaccurate and cumbersome. An alternative biochemical method is described and illustrated, which, it is claimed, is both more simple and more accurate. Slides of the thyroid and parathyroid are shown indicating that the histological criteria of function correlate with these biochemical findings during life. The classification of cases of pulmonary tuberculosis according to the mode of reaction rather than to the extent of lung invasion, is advocated. A terminology and the clinical, endocrine, and applied biochemical tests by which the groups can be recognized are described. Emphasis is laid on the necessity for research into the hereditary and environmental factors which lead to injury of the ductless glands, and hence lowered resistance to disease. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 14Fig. 15Fig. 16Fig. 17Fig. 18Fig. 19 PMID:19986232

Toxicity, Mutagenesis and Aging due to Endogenous Oxygen Radicals.

DTIC Science & Technology

1984-12-21

Fridovich, Investigations of the state of the -- manganese in Lactobacillus plantarum . Arch. Biochem. Biophys. 215, 589- 596 (1982). DiGuiseppi, J., and...4, 101-112 (1983). * Kono, Y., and I. Fridovich, Isolation and characterization of the * . pseudocatalase of Lactobacillus plantarum : a new manganese...Arch. Biochem. Biophys. 225, 640-648 (1983). Kono, Y., and I. Fridovich, The functional significance of manganese catalase in Lactobacillus plantarum

Adaptive sliding control of non-autonomous active suspension systems with time-varying loadings

NASA Astrophysics Data System (ADS)

Chen, Po-Chang; Huang, An-Chyau

2005-04-01

An adaptive sliding controller is proposed in this paper for controlling a non-autonomous quarter-car suspension system with time-varying loadings. The bound of the car-body loading is assumed to be available. Then, the reference coordinate is placed at the static position under the nominal loading so that the system dynamic equation is derived. Due to spring nonlinearities, the system property becomes asymmetric after coordinate transformation. Besides, in practical cases, system parameters are not easy to be obtained precisely for controller design. Therefore, in this paper, system uncertainties are lumped into two unknown time-varying functions. Since the variation bound of one of the unknown functions is not available, conventional adaptive schemes and robust designs are not applicable. To deal with this problem, the function approximation technique is employed to represent the unknown function as a finite combination of basis functions. The Lyapunov direct method can thus be used to find adaptive laws for updating coefficients in the approximating series and to prove stability of the closed-loop system. Since the position and velocity measurements of the unsprung mass are lumped into the unknown function, there is no need to install sensors on the axle and wheel assembly in the actual implementation. Simulation results are presented to show the performance of the proposed strategy.

Causal correlation of foliar biochemical concentrations with AVIRIS spectra using forced entry linear regression

NASA Technical Reports Server (NTRS)

Dawson, Terence P.; Curran, Paul J.; Kupiec, John A.

1995-01-01

A major goal of airborne imaging spectrometry is to estimate the biochemical composition of vegetation canopies from reflectance spectra. Remotely-sensed estimates of foliar biochemical concentrations of forests would provide valuable indicators of ecosystem function at regional and eventually global scales. Empirical research has shown a relationship exists between the amount of radiation reflected from absorption features and the concentration of given biochemicals in leaves and canopies (Matson et al., 1994, Johnson et al., 1994). A technique commonly used to determine which wavelengths have the strongest correlation with the biochemical of interest is unguided (stepwise) multiple regression. Wavelengths are entered into a multivariate regression equation, in their order of importance, each contributing to the reduction of the variance in the measured biochemical concentration. A significant problem with the use of stepwise regression for determining the correlation between biochemical concentration and spectra is that of 'overfitting' as there are significantly more wavebands than biochemical measurements. This could result in the selection of wavebands which may be more accurately attributable to noise or canopy effects. In addition, there is a real problem of collinearity in that the individual biochemical concentrations may covary. A strong correlation between the reflectance at a given wavelength and the concentration of a biochemical of interest, therefore, may be due to the effect of another biochemical which is closely related. Furthermore, it is not always possible to account for potentially suitable waveband omissions in the stepwise selection procedure. This concern about the suitability of stepwise regression has been identified and acknowledged in a number of recent studies (Wessman et al., 1988, Curran, 1989, Curran et al., 1992, Peterson and Hubbard, 1992, Martine and Aber, 1994, Kupiec, 1994). These studies have pointed to the lack of a physical link between wavelengths chosen by stepwise regression and the biochemical of interest, and this in turn has cast doubts on the use of imaging spectrometry for the estimation of foliar biochemical concentrations at sites distant from the training sites. To investigate this problem, an analysis was conducted on the variation in canopy biochemical concentrations and reflectance spectra using forced entry linear regression.

MUSIC-type imaging of small perfectly conducting cracks with an unknown frequency

NASA Astrophysics Data System (ADS)

Park, Won-Kwang

2015-09-01

MUltiple SIgnal Classification (MUSIC) is a famous non-iterative detection algorithm in inverse scattering problems. However, when the applied frequency is unknown, inaccurate locations are identified via MUSIC. This fact has been confirmed through numerical simulations. However, the reason behind this phenomenon has not been investigated theoretically. Motivated by this fact, we identify the structure of MUSIC-type imaging functionals with unknown frequency, by establishing a relationship with Bessel functions of order zero of the first kind. Through this, we can explain why inaccurate results appear.

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.

PubMed

Sen, Dilara; Keung, Albert J

2018-01-01

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus specificity by considering concentration, affinity, avidity, and sequestration effects.

Integration of electrochemistry in micro-total analysis systems for biochemical assays: recent developments.

PubMed

Xu, Xiaoli; Zhang, Song; Chen, Hui; Kong, Jilie

2009-11-15

Micro-total analysis systems (microTAS) integrate different analytical operations like sample preparation, separation and detection into a single microfabricated device. With the outstanding advantages of low cost, satisfactory analytical efficiency and flexibility in design, highly integrated and miniaturized devices from the concept of microTAS have gained widespread applications, especially in biochemical assays. Electrochemistry is shown to be quite compatible with microanalytical systems for biochemical assays, because of its attractive merits such as simplicity, rapidity, high sensitivity, reduced power consumption, and sample/reagent economy. This review presents recent developments in the integration of electrochemistry in microdevices for biochemical assays. Ingenious microelectrode design and fabrication methods, and versatility of electrochemical techniques are involved. Practical applications of such integrated microsystem in biochemical assays are focused on in situ analysis, point-of-care testing and portable devices. Electrochemical techniques are apparently suited to microsystems, since easy microfabrication of electrochemical elements and a high degree of integration with multi-analytical functions can be achieved at low cost. Such integrated microsystems will play an increasingly important role for analysis of small volume biochemical samples. Work is in progress toward new microdevice design and applications.

Evaluation method for the potential functionome harbored in the genome and metagenome

PubMed Central

2012-01-01

Background One of the main goals of genomic analysis is to elucidate the comprehensive functions (functionome) in individual organisms or a whole community in various environments. However, a standard evaluation method for discerning the functional potentials harbored within the genome or metagenome has not yet been established. We have developed a new evaluation method for the potential functionome, based on the completion ratio of Kyoto Encyclopedia of Genes and Genomes (KEGG) functional modules. Results Distribution of the completion ratio of the KEGG functional modules in 768 prokaryotic species varied greatly with the kind of module, and all modules primarily fell into 4 patterns (universal, restricted, diversified and non-prokaryotic modules), indicating the universal and unique nature of each module, and also the versatility of the KEGG Orthology (KO) identifiers mapped to each one. The module completion ratio in 8 phenotypically different bacilli revealed that some modules were shared only in phenotypically similar species. Metagenomes of human gut microbiomes from 13 healthy individuals previously determined by the Sanger method were analyzed based on the module completion ratio. Results led to new discoveries in the nutritional preferences of gut microbes, believed to be one of the mutualistic representations of gut microbiomes to avoid nutritional competition with the host. Conclusions The method developed in this study could characterize the functionome harbored in genomes and metagenomes. As this method also provided taxonomical information from KEGG modules as well as the gene hosts constructing the modules, interpretation of completion profiles was simplified and we could identify the complementarity between biochemical functions in human hosts and the nutritional preferences in human gut microbiomes. Thus, our method has the potential to be a powerful tool for comparative functional analysis in genomics and metagenomics, able to target unknown environments containing various uncultivable microbes within unidentified phyla. PMID:23234305

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation*

PubMed Central

Netz, Daili J. A.; Pierik, Antonio J.; Stümpfig, Martin; Bill, Eckhard; Sharma, Anil K.; Pallesen, Leif J.; Walden, William E.; Lill, Roland

2012-01-01

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo. PMID:22362766

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

PubMed

Armas, Pablo; Agüero, Tristán H; Borgognone, Mariana; Aybar, Manuel J; Calcaterra, Nora B

2008-10-17

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces.

PubMed

Boys, Alexander J; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J; Estroff, Lara A

2017-09-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors.

Next Generation Tissue Engineering of Orthopedic Soft Tissue-to-Bone Interfaces

PubMed Central

Boys, Alexander J.; McCorry, Mary Clare; Rodeo, Scott; Bonassar, Lawrence J.; Estroff, Lara A.

2017-01-01

Soft tissue-to-bone interfaces are complex structures that consist of gradients of extracellular matrix materials, cell phenotypes, and biochemical signals. These interfaces, called entheses for ligaments, tendons, and the meniscus, are crucial to joint function, transferring mechanical loads and stabilizing orthopedic joints. When injuries occur to connected soft tissue, the enthesis must be re-established to restore function, but due to structural complexity, repair has proven challenging. Tissue engineering offers a promising solution for regenerating these tissues. This prospective review discusses methodologies for tissue engineering the enthesis, outlined in three key design inputs: materials processing methods, cellular contributions, and biochemical factors. PMID:29333332

Biology and clinical relevance of noncoding sno/scaRNAs.

PubMed

Cao, Thuy; Rajasingh, Sheeja; Samanta, Saheli; Dawn, Buddhadeb; Bittel, Douglas C; Rajasingh, Johnson

2018-02-01

Small nucleolar RNAs (snoRNAs) are a group of noncoding RNAs that perform various biological functions, including biochemical modifications of other RNAs, precursors of miRNA, splicing, and telomerase activity. The small Cajal body-associated RNAs (scaRNAs) are a subset of the snoRNA family and collect in the Cajal body where they perform their canonical function to biochemically modify spliceosomal RNAs prior to maturation. Failure of sno/scaRNAs have been implicated in pathology such as congenital heart anomalies, neuromuscular disorders, and various malignancies. Thus, understanding of sno/scaRNAs demonstrates the clinical value. Copyright © 2018 Elsevier Inc. All rights reserved.

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

PubMed

Findley, Seth D; Mormile, Melanie R; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H; Kerley, Monty; Stacey, Gary

2011-11-01

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

A grid layout algorithm for automatic drawing of biochemical networks.

PubMed

Li, Weijiang; Kurata, Hiroyuki

2005-05-01

Visualization is indispensable in the research of complex biochemical networks. Available graph layout algorithms are not adequate for satisfactorily drawing such networks. New methods are required to visualize automatically the topological architectures and facilitate the understanding of the functions of the networks. We propose a novel layout algorithm to draw complex biochemical networks. A network is modeled as a system of interacting nodes on squared grids. A discrete cost function between each node pair is designed based on the topological relation and the geometric positions of the two nodes. The layouts are produced by minimizing the total cost. We design a fast algorithm to minimize the discrete cost function, by which candidate layouts can be produced efficiently. A simulated annealing procedure is used to choose better candidates. Our algorithm demonstrates its ability to exhibit cluster structures clearly in relatively compact layout areas without any prior knowledge. We developed Windows software to implement the algorithm for CADLIVE. All materials can be freely downloaded from http://kurata21.bio.kyutech.ac.jp/grid/grid_layout.htm; http://www.cadlive.jp/ http://kurata21.bio.kyutech.ac.jp/grid/grid_layout.htm; http://www.cadlive.jp/

[Simulation of vegetation indices optimizing under retrieval of vegetation biochemical parameters based on PROSPECT + SAIL model].

PubMed

Wu, Ling; Liu, Xiang-Nan; Zhou, Bo-Tian; Liu, Chuan-Hao; Li, Lu-Feng

2012-12-01

This study analyzed the sensitivities of three vegetation biochemical parameters [chlorophyll content (Cab), leaf water content (Cw), and leaf area index (LAI)] to the changes of canopy reflectance, with the effects of each parameter on the wavelength regions of canopy reflectance considered, and selected three vegetation indices as the optimization comparison targets of cost function. Then, the Cab, Cw, and LAI were estimated, based on the particle swarm optimization algorithm and PROSPECT + SAIL model. The results showed that retrieval efficiency with vegetation indices as the optimization comparison targets of cost function was better than that with all spectral reflectance. The correlation coefficients (R2) between the measured and estimated values of Cab, Cw, and LAI were 90.8%, 95.7%, and 99.7%, and the root mean square errors of Cab, Cw, and LAI were 4.73 microg x cm(-2), 0.001 g x cm(-2), and 0.08, respectively. It was suggested that to adopt vegetation indices as the optimization comparison targets of cost function could effectively improve the efficiency and precision of the retrieval of biochemical parameters based on PROSPECT + SAIL model.

Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster.

PubMed

Okada, Hirokazu; Ebhardt, H Alexander; Vonesch, Sibylle Chantal; Aebersold, Ruedi; Hafen, Ernst

2016-09-01

The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations.

Investigation on energy conversion technology using biochemical reaction elements, 2

NASA Astrophysics Data System (ADS)

1994-03-01

For measures taken for resource/energy and environmental issues, a study is made on utilization of microbial biochemical reaction. As a reaction system using chemical energy, cited is production of petroleum substitution substances and food/feed by CO2 fixation using hydrogen energy and hydrogen bacteria. As to photo energy utilization, regarded as promising are CO2 fixation using photo energy and microalgae, and production of hydrogen and useful carbon compound using photosynthetic organisms. As living organism/electric energy interconversion, cited is the culture of chemoautotrophic bacteria which fix CO2 using electric energy. For enhancing its conversion efficiency, it is important to develop a technology of gene manipulation of the bacteria and a system to use functional biochemical elements adaptable to the electrode reaction. With regard to utilization of the microorganism metabolic function, the paper presents emission of soluble nitrogen in the hydrosphere into the atmosphere using denitrifying bacteria, removal of phosphorus, reduction in environmental pollution caused by heavy metal dilute solutions, and recovery as resources, etc.

[Primary biliary cirrhosis-autoimmune hepatitis overlap syndrome. Contribution of two new cases].

PubMed

Fernández Fernández, F J; de la Fuente Aguado, J; Pérez Fernández, S; Sopeña Pérez-Argüelles, B; Nodar Germiñas, A; Butrón Vila, M

2005-03-01

The autoimmune hepatitis (AIH)-primary biliary cirrhosis (PBC) overlap syndrome is characterized for clinical, biochemical, immunological, and histological features overlapping those of AIH and PBC, whose pathogenesis and more appropriate treatment are unknown at present. We describe two new patients of this entity, which made debut with cholestasic acute hepatitis accompanied of hypergammaglobulinemia. In the first patient was demonstrated the presence of AMA, ASMA, and anti-LKM1 autoantibodies; and ANA in the second one. The histological findings showed changes suggestive of AIH and PBC. After the start of immunosuppressive treatment, associated to ursodeoxycholic acid in one patient, a successful outcome was observed.

Massive Submandibular Sialolith: Complete Radiographic Registration and Biochemical Analysis through X-Ray Diffraction

PubMed Central

de Carvalho Mattos, Mayara Jessica; Ferrari, Francine; dos Reis Neto, José Manoel; Carta Gambus, Luiz Carlos; Couto Souza, Paulo Henrique; Berti-Couto, Soraya de Azambuja

2014-01-01

Sialolithiasis is a pathologic condition that affects 60 million people per year, which is caused by the presence of calcified structures, named sialoliths, inside the salivary glands and their salivary ducts. Despite the large incidence of sialolithiasis, its etiology is still unknown. In the present case report, a 47-year-old female patient, presenting with local pain and hampered mouth opening, underwent a surgical approach for the removal of a 20 mm sialolith, which was further analyzed through X-ray diffraction. In parallel, a radiographic registration of 8 years, covering all the period for sialolith formation, is presented along the case report. PMID:25258693

Massive Submandibular Sialolith: Complete Radiographic Registration and Biochemical Analysis through X-Ray Diffraction.

PubMed

Franco, Ademir; de Carvalho Mattos, Mayara Jessica; Ferrari, Francine; Dos Reis Neto, José Manoel; Carta Gambus, Luiz Carlos; Couto Souza, Paulo Henrique; Berti-Couto, Soraya de Azambuja

2014-01-01

Sialolithiasis is a pathologic condition that affects 60 million people per year, which is caused by the presence of calcified structures, named sialoliths, inside the salivary glands and their salivary ducts. Despite the large incidence of sialolithiasis, its etiology is still unknown. In the present case report, a 47-year-old female patient, presenting with local pain and hampered mouth opening, underwent a surgical approach for the removal of a 20 mm sialolith, which was further analyzed through X-ray diffraction. In parallel, a radiographic registration of 8 years, covering all the period for sialolith formation, is presented along the case report.

Atomic structures of corkscrew-forming segments of SOD1 reveal varied oligomer conformations.

PubMed

Sangwan, Smriti; Sawaya, Michael R; Murray, Kevin A; Hughes, Michael P; Eisenberg, David S

2018-02-17

The aggregation cascade of disease-related amyloidogenic proteins, terminating in insoluble amyloid fibrils, involves intermediate oligomeric states. The structural and biochemical details of these oligomers have been largely unknown. Here we report crystal structures of variants of the cytotoxic oligomer-forming segment residues 28-38 of the ALS-linked protein, SOD1. The crystal structures reveal three different architectures: corkscrew oligomeric structure, nontwisting curved sheet structure and a steric zipper proto-filament structure. Our work highlights the polymorphism of the segment 28-38 of SOD1 and identifies the molecular features of amyloidogenic entities. © 2018 The Protein Society.

A Systematic Protocol for the Characterization of Hsp90 Modulators

PubMed Central

Matts, Robert L.; Brandt, Gary E. L.; Lu, Yuanming; Dixit, Anshuman; Mollapour, Mehdi; Wang, Suiquan; Donnelly, Alison C.; Neckers, Leonard; Verkhivker, Gennady; Blagg, Brian S. J.

2015-01-01

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA. PMID:21129982

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

Effect of carprofen on hemostatic variables in dogs.

PubMed

Hickford, F H; Barr, S C; Erb, H N

2001-10-01

To evaluate the effect of carprofen on hemostatic variables in clinically normal dogs. 12 clinically normal Labrador Retrievers. 10 dogs (6 females, 4 males) received carprofen (2.2 mg/kg of body weight, PO, q 12 h) for 5 days. Two dogs (untreated control group; 1 female, 1 male) did not receive carprofen. Hemostatic variables (platelet count, activated partial thromboplastin time, prothrombin time, fibrinogen, platelet aggregation, and bleeding time) were assessed for all dogs prior to treatment, on day 5 of treatment, and 2 and 7 days after discontinuation of the drug (days 7 and 12). Serum biochemical variables and Hct were assessed prior to treatment and on days 5 and 12. In dogs receiving carprofen, platelet aggregation was significantly decreased, and onset of aggregation was significantly delayed on days 5, 7, and 12, compared with pretreatment values. Activated partial thromboplastin time was significantly increased on days 5, 7, and 12 over pretreatment values in treated dogs, but values remained within reference ranges. Significant differences were not detected in buccal mucosal bleeding time, other serum biochemical and hemostatic variables, or Hct, compared with pretreatment values and the internal control group. Administration of carprofen for 5 days causes minor but not clinically important alterations in hemostatic and serum biochemical variables in clinically normal Labrador Retrievers. Carprofen is commonly used to treat osteoarthritis and chronic pain in dogs, but prior to this study, its effect on platelet aggregation and hemostatic variables was unknown.

Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

PubMed Central

Nam, Ju-Suk; Chakraborty, Chiranjib; Sharma, Ashish Ranjan; Her, Young; Bae, Kee-Jeong; Sharma, Garima; Doss, George Priya; Lee, Sang-Soo; Hong, Myung-Sun; Song, Dong-Keun

2014-01-01

Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers. PMID:24686518

Biochemical Association of Metabolic Profile and Microbiome in Chronic Pressure Ulcer Wounds

PubMed Central

Ammons, Mary Cloud B.; Morrissey, Kathryn; Tripet, Brian P.; Van Leuven, James T.; Han, Anne; Lazarus, Gerald S.; Zenilman, Jonathan M.; Stewart, Philip S.; James, Garth A.; Copié, Valérie

2015-01-01

Chronic, non-healing wounds contribute significantly to the suffering of patients with co-morbidities in the clinical population with mild to severely compromised immune systems. Normal wound healing proceeds through a well-described process. However, in chronic wounds this process seems to become dysregulated at the transition between resolution of inflammation and re-epithelialization. Bioburden in the form of colonizing bacteria is a major contributor to the delayed headlining in chronic wounds such as pressure ulcers. However how the microbiome influences the wound metabolic landscape is unknown. Here, we have used a Systems Biology approach to determine the biochemical associations between the taxonomic and metabolomic profiles of wounds colonized by bacteria. Pressure ulcer biopsies were harvested from primary chronic wounds and bisected into top and bottom sections prior to analysis of microbiome by pyrosequencing and analysis of metabolome using 1H nuclear magnetic resonance (NMR) spectroscopy. Bacterial taxonomy revealed that wounds were colonized predominantly by three main phyla, but differed significantly at the genus level. While taxonomic profiles demonstrated significant variability between wounds, metabolic profiles shared significant similarity based on the depth of the wound biopsy. Biochemical association between taxonomy and metabolic landscape indicated significant wound-to-wound similarity in metabolite enrichment sets and metabolic pathway impacts, especially with regard to amino acid metabolism. To our knowledge, this is the first demonstration of a statistically robust correlation between bacterial colonization and metabolic landscape within the chronic wound environment. PMID:25978400

Elastin-like Protein-Hyaluronic acid (ELP-HA) Hydrogels with Decoupled Mechanical and Biochemical cues for Cartilage Regeneration

PubMed Central

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C.; Yang, Fan

2018-01-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows varyiation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. PMID:28268018

Effect of deuterium-depleted water on selected cardiometabolic parameters in fructose-treated rats.

PubMed

Rehakova, R; Klimentova, J; Cebova, M; Barta, A; Matuskova, Z; Labas, P; Pechanova, O

2016-10-24

Deuterium-depleted water (DDW) has a lower concentration of deuterium than occurs naturally (less than 145 ppm). While effects of DDW on cancer started to be intensively studied, the effects on cardiovascular system are completely unknown. Thus, we aimed to analyze the effects of DDW (55+/-5 ppm) administration to 12-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) treated with 15 % fructose for 6 weeks. Blood pressure (BP) and selected biochemical parameters were measured together with determination of nitric oxide synthase (NOS) activity and iNOS and eNOS protein expressions in the left ventricle (LV) and aorta. Neither DDW nor fructose had any significant effect on BP in both strains. DDW treatment decreased total cholesterol and triglyceride levels in WKY, but it was not able to prevent increase in the same parameters elevated due to fructose treatment in SHR. Both fructose and DDW increased insulin level in WKY. Fructose did not affect NOS activity either in WKY or SHR. DDW increased NOS activity in LV of both WKY and SHR, while it decreased NOS activity and iNOS expression in the aorta of SHR with or without fructose treatment. In conclusion, DDW treatment significantly modified biochemical parameters in WKY together with NOS activity elevation in the heart. On the other hand, it did not affect biochemical parameters in SHR, but decreased NOS activity elevated due to iNOS upregulation in the aorta.

Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.

PubMed

Zhu, Danqing; Wang, Huiyuan; Trinh, Pavin; Heilshorn, Sarah C; Yang, Fan

2017-05-01

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Roles for Msx and Dlx homeoproteins in vertebrate development.

PubMed

Bendall, A J; Abate-Shen, C

2000-04-18

This review provides a comparative analysis of the expression patterns, functions, and biochemical properties of Msx and Dlx homeobox genes. These comprise multi-gene families that are closely related with respect to sequence features as well as expression patterns during vertebrate development. Thus, members of the Msx and Dlx families are expressed in overlapping, but distinct, patterns and display complementary or antagonistic functions, depending upon the context. A common theme shared among Msx and Dlx genes is that they are required during early, middle, and late phases of development where their differential expression mediates patterning, morphogenesis, and histogenesis of tissues in which they are expressed. With respect to their biochemical properties, Msx proteins function as transcriptional repressors, while Dlx proteins are transcriptional activators. Moreover, their ability to oppose each other's transcriptional actions implies a mechanism underlying their complementary or antagonistic functions during development.

A new polytopic approach for the unknown input functional observer design

NASA Astrophysics Data System (ADS)

Bezzaoucha, Souad; Voos, Holger; Darouach, Mohamed

2018-03-01

In this paper, a constructive procedure to design Functional Unknown Input Observers for nonlinear continuous time systems is proposed under the Polytopic Takagi-Sugeno framework. An equivalent representation for the nonlinear model is achieved using the sector nonlinearity transformation. Applying the Lyapunov theory and the ? attenuation, linear matrix inequalities conditions are deduced which are solved for feasibility to obtain the observer design matrices. To cope with the effect of unknown inputs, classical approach of decoupling the unknown input for the linear case is used. Both algebraic and solver-based solutions are proposed (relaxed conditions). Necessary and sufficient conditions for the existence of the functional polytopic observer are given. For both approaches, the general and particular cases (measurable premise variables, full state estimation with full and reduced order cases) are considered and it is shown that the proposed conditions correspond to the one presented for standard linear case. To illustrate the proposed theoretical results, detailed numerical simulations are presented for a Quadrotor Aerial Robots Landing and a Waste Water Treatment Plant. Both systems are highly nonlinear and represented in a T-S polytopic form with unmeasurable premise variables and unknown inputs.

Testing the Hypothesis of a Homoscedastic Error Term in Simple, Nonparametric Regression

ERIC Educational Resources Information Center

Wilcox, Rand R.

2006-01-01

Consider the nonparametric regression model Y = m(X)+ [tau](X)[epsilon], where X and [epsilon] are independent random variables, [epsilon] has a median of zero and variance [sigma][squared], [tau] is some unknown function used to model heteroscedasticity, and m(X) is an unknown function reflecting some conditional measure of location associated…

Efficient Learning Algorithms with Limited Information

ERIC Educational Resources Information Center

De, Anindya

2013-01-01

The thesis explores efficient learning algorithms in settings which are more restrictive than the PAC model of learning (Valiant) in one of the following two senses: (i) The learning algorithm has a very weak access to the unknown function, as in, it does not get labeled samples for the unknown function (ii) The error guarantee required from the…

Improved mapping of radio sources from VLBI data by least-square fit

NASA Technical Reports Server (NTRS)

Rodemich, E. R.

1985-01-01

A method is described for producing improved mapping of radio sources from Very Long Base Interferometry (VLBI) data. The method described is more direct than existing Fourier methods, is often more accurate, and runs at least as fast. The visibility data is modeled here, as in existing methods, as a function of the unknown brightness distribution and the unknown antenna gains and phases. These unknowns are chosen so that the resulting function values are as near as possible to the observed values. If researchers use the radio mapping source deviation to measure the closeness of this fit to the observed values, they are led to the problem of minimizing a certain function of all the unknown parameters. This minimization problem cannot be solved directly, but it can be attacked by iterative methods which we show converge automatically to the minimum with no user intervention. The resulting brightness distribution will furnish the best fit to the data among all brightness distributions of given resolution.

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction.

PubMed

Varela, Ana T; Gomes, Ana P; Simões, Anabela M; Teodoro, João S; Duarte, Filipe V; Rolo, Anabela P; Palmeira, Carlos M

2008-12-01

Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

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

Varela, Ana T.; Gomes, Ana P.; Simoes, Anabela M.

2008-12-01

Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10{mu}M, significantly depressesmore » the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25{mu}M stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.« less

Simultaneous ablation of prmt-1 and prmt-5 abolishes asymmetric and symmetric arginine dimethylations in Caenorhabditis elegans.

PubMed

Hirota, Keiko; Shigekawa, Chihiro; Araoi, Sho; Sha, Liang; Inagawa, Takayuki; Kanou, Akihiko; Kako, Koichiro; Daitoku, Hiroaki; Fukamizu, Akiyoshi

2017-06-01

Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

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

Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65more » Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).« less

EBS7 is a plant-specific component of a highly conserved endoplasmic reticulum-associated degradation system in Arabidopsis

PubMed Central

Liu, Yidan; Zhang, Congcong; Wang, Dinghe; Su, Wei; Liu, Linchuan; Wang, Muyang; Li, Jianming

2015-01-01

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an essential part of an ER-localized protein quality-control system for eliminating terminally misfolded proteins. Recent studies have demonstrated that the ERAD machinery is conserved among yeast, animals, and plants; however, it remains unknown if the plant ERAD system involves plant-specific components. Here we report that the Arabidopsis ethyl methanesulfonate-mutagenized brassinosteroid-insensitive 1 suppressor 7 (EBS7) gene encodes an ER membrane-localized ERAD component that is highly conserved in land plants. Loss-of-function ebs7 mutations prevent ERAD of brassinosteroid insensitive 1-9 (bri1-9) and bri1-5, two ER-retained mutant variants of the cell-surface receptor for brassinosteroids (BRs). As a result, the two mutant receptors accumulate in the ER and consequently leak to the plasma membrane, resulting in the restoration of BR sensitivity and phenotypic suppression of the bri1-9 and bri1-5 mutants. EBS7 accumulates under ER stress, and its mutations lead to hypersensitivity to ER and salt stresses. EBS7 interacts with the ER membrane-anchored ubiquitin ligase Arabidopsis thaliana HMG-CoA reductase degradation 1a (AtHrd1a), one of the central components of the Arabidopsis ERAD machinery, and an ebs7 mutation destabilizes AtHrd1a to reduce polyubiquitination of bri1-9. Taken together, our results uncover a plant-specific component of a plant ERAD pathway and also suggest its likely biochemical function. PMID:26371323

Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1

PubMed Central

Capellini, Terence D.; Vaccari, Giulia; Ferretti, Elisabetta; Fantini, Sebastian; He, Mu; Pellegrini, Massimo; Quintana, Laura; Di Giacomo, Giuseppina; Sharpe, James; Selleri, Licia; Zappavigna, Vincenzo

2010-01-01

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development. PMID:20627960

Running wheel exercise reduces α-synuclein aggregation and improves motor and cognitive function in a transgenic mouse model of Parkinson's disease

PubMed Central

Barkow, Jessica Cummiskey; Freed, Curt R.

2017-01-01

Exercise has been recommended to improve motor function in Parkinson patients, but its value in altering progression of disease is unknown. In this study, we examined the neuroprotective effects of running wheel exercise in mice. In adult wild-type mice, one week of running wheel activity led to significantly increased DJ-1 protein concentrations in muscle and plasma. In DJ-1 knockout mice, running wheel performance was much slower and Rotarod performance was reduced, suggesting that DJ-1 protein is required for normal motor activity. To see if exercise can prevent abnormal protein deposition and behavioral decline in transgenic animals expressing a mutant human form of α-synuclein in all neurons, we set up running wheels in the cages of pre-symptomatic animals at 12 months old. Activity was monitored for a 3-month period. After 3 months, motor and cognitive performance on the Rotarod and Morris Water Maze were significantly better in running animals compared to control transgenic animals with locked running wheels. Biochemical analysis revealed that running mice had significantly higher DJ-1, Hsp70 and BDNF concentrations and had significantly less α-synuclein aggregation in brain compared to control mice. By contrast, plasma concentrations of α-synuclein were significantly higher in exercising mice compared to control mice. Our results suggest that exercise may slow the progression of Parkinson’s disease by preventing abnormal protein aggregation in brain. PMID:29272304

Functional and Evolutionary Characterization of a Gene Transfer Agent’s Multilocus “Genome”

PubMed Central

Hynes, Alexander P.; Shakya, Migun; Mercer, Ryan G.; Grüll, Marc P.; Bown, Luke; Davidson, Fraser; Steffen, Ekaterina; Matchem, Heidi; Peach, Mandy E.; Berger, Tim; Grebe, Katherine; Zhaxybayeva, Olga; Lang, Andrew S.

2016-01-01

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell’s genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures “repurposed” by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA “genome” consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA “genome” loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA “genome” is presumably a result of selection on the host organism to retain GTA functionality. PMID:27343288

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation

PubMed Central

Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

2014-01-01

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-l-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., 1994), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis. PMID:24286468

Dynamics of biochemical processes and redox conditions in geochemically linked landscapes of oligotrophic bogs

NASA Astrophysics Data System (ADS)

Inisheva, L. I.; Szajdak, L.; Sergeeva, M. A.

2016-04-01

The biological activity in oligotrophic peatlands at the margins of the Vasyugan Mire has been studied. It is shown found that differently directed biochemical processes manifest themselves in the entire peat profile down to the underlying mineral substrate. Their activity is highly variable. It is argued that the notion about active and inert layers in peat soils is only applicable for the description of their water regime. The degree of the biochemical activity is specified by the physical soil properties. As a result of the biochemical processes, a micromosaic aerobic-anaerobic medium is developed under the surface waterlogged layer of peat deposits. This layer contains the gas phase, including oxygen. It is concluded that the organic and mineral parts of peat bogs represent a single functional system of a genetic peat profile with a clear record of the history of its development.

Super-resolution links vinculin localization to function in focal adhesions.

PubMed

Giannone, Grégory

2015-07-01

Integrin-based focal adhesions integrate biochemical and biomechanical signals from the extracellular matrix and the actin cytoskeleton. The combination of three-dimensional super-resolution imaging and loss- or gain-of-function protein mutants now links the nanoscale dynamic localization of proteins to their activation and function within focal adhesions.

A deterministic global optimization using smooth diagonal auxiliary functions

NASA Astrophysics Data System (ADS)

Sergeyev, Yaroslav D.; Kvasov, Dmitri E.

2015-04-01

In many practical decision-making problems it happens that functions involved in optimization process are black-box with unknown analytical representations and hard to evaluate. In this paper, a global optimization problem is considered where both the goal function f (x) and its gradient f‧ (x) are black-box functions. It is supposed that f‧ (x) satisfies the Lipschitz condition over the search hyperinterval with an unknown Lipschitz constant K. A new deterministic 'Divide-the-Best' algorithm based on efficient diagonal partitions and smooth auxiliary functions is proposed in its basic version, its convergence conditions are studied and numerical experiments executed on eight hundred test functions are presented.